Methods for diagnosing and treating autoimmune disease

ABSTRACT

The invention provides a method of detecting autoimmune disease in a mammal, comprising providing a biological sample from a mammal and detecting proteasome activity, wherein a reduction in proteasome activity from a basal state is indicative of autoimmune disease. In addition, the invention encompasses a method of treating an autoimmune disease in a mammal, comprising administering to a mammal suspected of suffering from an autoimmune disease an agent which restores NFκB activity in an amount and for a time sufficient to result in normal NFκB activity in the mammal.

This is a continuation-in-part of application Ser. No. 09/031,629, filedFeb. 27, 1998 now U.S. Pat. No. 6,617,171.

FIELD OF THE INVENTION

The invention relates in general to the diagnosis and treatment ofimmune disorders.

BACKGROUND OF THE INVENTION Proteolysis in the Cell A. The Proteasome

In the cytosol, there is a soluble proteolytic pathway that requires ATPand involves covalent conjugation of the cellular proteins with thesmall polypeptide ubiquitin, or Ub, (Hershko et al., 1992, Ann. Rev.Biochem., 61: 761-807; Rechsteiner et al., 1987, Ann. Rev. Cell. Biol.,3: 1-30). Therafer, the conjugated proteins are hydrolyzed by a 26Sproteolytic complex containing a 20S degradative particle called theproteasome (Goldberg, 1992, Eur. J. Biochem., 203: 9-23); Goldberg etal., 1992, Nature, 357: 375-379). This multicomponent system is known tocatalyze the selective degradation of highly abnormal proteins andshort-lived regulatory proteins. However, the system also appears to beresponsible for the breakdown of most proteins in maturing reticulocytes(Boches et al., 1982, Science, 215: 978-980); Spenser et al., 1985, J.Biol. Chem., 257: 14122-14127), in growing fibroblasts (Ciechanover etal., 1984, Cell, 37: 57-66; Gronostajski et al., 1985, J. Biol. Chem,260: 3344-3349) and in atrophying skeletal muscle.

The first step in degradation of many proteins involves theirconjugation to Ub by an ATP-requiring process, as described below. Theubiquitinated proteins are then degraded by an ATP-dependent proteolyticcomplex, referred to above, known as the 26S proteasome complex.

The precise nature of the 26S proteasome complex is unclear, although ithas been shown that the 1060-1500 kDa (26S) complex can be formed inextracts of energy-depleted reticulocytes by an ATP-dependentassociation of three components, refereed to as OF-1, OF-2, and OF-3(Ganoth et al., 1988, J. Biol. Chem., 263; 12412-12419). A large (˜700kDa) multimeric protease found in the cytoplasm and nucleus ofeukaryotic cells, referred to as the proteasome, is a component (OF-3)(Driscoll et al., 1992, J. Biol. Chem., 265: 4789-4792; Eytan et al.,1989, Proc. Natl. Acad. Sci. U.S.A., 86: 7751-5 7755; Orlowski et al.,1990, Biochemistry, 29: 10289-10297; Rivet, 1989, Arch. Biochem. Biophys., 268: 1-8).

The proteasome is believed to make up the catalytic core of the large26S multisubunit cytoplasmic particle necessary for theubiquitin-dependent pathway of intracellular proteolysis (Driscoll etal., 1990, J. Biol., Chem., 265: 4789-4692; Eytan et al., 1989, Proc.Natl. Acad. Sci. U.S.A., 86: 7751-7755; Hough et al., 1987,Biochemistry, 262: 8303-8313; McGuire et al., 1988, Biochim. Biophys.Acta., 967: 195-203; Rechsteiner et al., 1987, Ann. Rev. Cell. Biol., 3:1-30; Waxman et al., 1987, J. Biol. Chem., 262: 2451-2457). By itself,the proteasome is unable to degrade ubiquitinated proteins, but providesmost of the proteolytic activity of the 26S proteasome complex.

There is another ATP-dependent protease that is involved in degradationof ubiquitinated proteins, forms a complex with the proteasome andappears to be part of the 26S proteasome complex, which rapidly degradesproteins conjugated to ubiquitin. This protease, referred to asmultipain, has been identified in muscle and plays an essential role inthe ATP/ubiquitin-dependent pathway.

The complex formed between multipain and proteasome in vitro appearsvery similar or identical to the 1500 kDa Ub-conjugate, degradingenzyme, or 26S proteolytic complex, isolated from reticulocytes andmuscle. The complexes contain the characteristic 20-30 kDa proteasomesubunits, plus a number of larger subunits, including the six largepolypeptides found in multipain. The complex formed contains at least10-12 polypeptides of 40-150 kDa. A 40 kDa polypeptide regulator of theproteasome, which inhibits the proteasome's proteolytic activities hasbeen purified from reticulocytes and shown to be an ATP-binding proteinwhose release appears to activate proteolysis. The isolated regulatorexists as a 250 kDa multimer and is quite labile (at 42° C.). It can bestabilized by the addition of ATP or a nonhydrolyzable ATP analog,although the purified regulator does not require ATP to inhibitproteasome function and lacks ATPase activity. The regulator has beenshown to correspond to an essential component of the 1500 kDaproteolytic complex. The regulator appears identical to CF-2 by manycriteria. These findings suggest that the regulator plays a role in theATP—dependent mechanism of the 26S proteasome complex.

The 20S proteasome is composed of about 15 distinct 20-30 kDa subunits.It contains at least three different peptidases that cleave specificallyan the carboxyl side of the hydrophobic, basic, and acidic amino acids(Goldberg et al., 1992, Nature, 357: 375-379: Goldberg, 1992, Eur. J.Biochem., 203: 9-23; Orlowski, 1990, Biochemitry, 29: 10289-10297;Rivett et al., 1989, Arch. Biochem. Biophys., 218: 1; Rivett et al.,1989, J. Biol. Chem., 264: 12215-12219;Tanaka et al., 1992, New Biol. 4:1-11). These peptidases are referred to as the chymotrypsin-likepeptidase, the trypsin-like peptidase, and the peptidylglutamylpeptidase. Which subunits are responsible for these activities isunknown although the cDNA's encoding several subunits have been cloned(Tanaka et al., 1992, New Biol., 4: 1-11).

B. Ubiqiuitination and Phosphorylation in Protein Processing

As reviewed by Hopkin (1997, J. NIH Research, 9: 36-42) and brieflysummarized herein, insight into the mechanisms by which proteolysis iscontrolled come from studies of the eukaryotic cell cycle. To proceedthrough the cell cycle, replicating its genome and dividing theresulting DNA between daughter cells during mitosis, a cell mustappropriately activate and inactivate the regulators of cell division,the cyclin-dependent kinases (Cdks). To control Cdks, cells canspecifically degrade the cyclin proteins that activate Cdks and theinhibitors that inactivate them. One mechanism by which specificity intargeted proteolysis is achieved is ubiquitination, the process by whichcells tack long chains of a 76-amino acid marker protein calledubiquitin (Ub) onto proteins that are destined for destruction.Ubiquitination of a handful of cyclins and Cdk inhibitors leads to theirtimely demise and allows a cell to complete mitosis or to replicate itsDNA; further, it is believed that phosphorylation of unstable proteins,such as the cyclins, often increases their susceptibility toubiquitination and subsequent elimination.

As described below, ubiquitination affects signal transduction, as itmay mark certain cell-surface growth-factor receptors for endocytosisand destruction; further, it is known that ubiquitination, coupled withphosphorylation, stimulates the signaling pathway that activates thetranscription factor NFκB. Ubiquitin also plays a role in proteindegradation pathways regulating cell differentiation and death duringdevelopment

Ubiquitination and the Cell Cycle

Evidence that ubiquitination was interesting from the point of view ofregulation came with the development of a mouse cell line that arrestsin the G², or gap 2, phase of the cell cycle; these cells harbor adefect that cripples an enzyme that activates Ub before it can bind toproteins, such as the cyclins, that must be targeted for destruction.Prior to this work, ubiquitination was viewed only as a means foreliminating damaged, denatured, and misfolded proteins.

Most of the proteolysis that occurs in cells involves the degradation ofUb-conjugated proteins. As stated above, the proteasome recognizes thepolyubiquitin tag, selectively admits proteins to which this marker iscomplexed and then cleaves them into small peptide fragments.Ubiquitination is dependent upon a series of proteins named for theirorder of elution from a Ub-affinity column. Ub-activating enzymes,called Els, prime Ub for transfer to a substrate protein by forming atemporary thioester linkage between a terminal glycine of Ub and one oftheir own cysteine residues. Enter the Ub-conjugating proteinsgenerically called E2s. These enzymes accept activated Ub from an E1 andtransfer it to the substrate protein, either directly or with the helpof a Ub-ligase protein, or E3; interactions between different E2s andE3s may contribute to the substrate specificity of the ubiquitinationreaction. Yeast maintain a cadre of more than a dozen structurallyrelated E2s as well as a handful of E3s (reviewed by Haas and Siepmann,1997, FASEB J., 11: 1257-1268). Functional homologues of these proteinshave been found in humans (see Honda et al., 1997, FEBS Lett., 420:25-27).

Even within the cell cycle, different sets of E2s and E3s function tomark cyclins and Cdk inhibitors for destruction. Together these proteinsregulate entry into new cycles of cell division, initiation of DNAreplication, and the onset of mitosis. In yeast, cyclins bind to andactivate Cdc28, which then pushes cells into the next phase of the cellcycle, initiating cell division. It is said that the concentrations ofboth the cyclins and the Cdk inhibitors that drive the cell cyclethrough their interactions with Cdc28 may be tightly controlled byUb-associated proteolysis. The G1 cyclins Cln1, Cln2, and Cln3 activateCdc28, by which they are then reciprocally phosphorylated; thisphosphorylation marks the cyclins for ubiquitination and subsequentdestruction by the proteasome.

The Ub-ligase complex that ubiquitinates the cell-cycle proteins thatcontrol the completion of mitosis is known to be activated byphosphorylation. The coupling of cyclin B and its kinase Cdc2 initiatesmitosis in yeast. In that system, cyclin B accumulates during interphaseuntil its pairing with Cdc2 drives the cell into mitosis and leads toits eventual destruction. The cyclosome (also called theanaphase-promoting complex , or APC), a 20S nuclear particle whichserves as the Ub-ligase complex, helps to ubiquitinate the mitoticcyclins A and B as well as the as-yet-unidentified “glue” proteins thatbind sister chromatids together during metaphase. Late in mitosis, anunknown kinase phosphorylates and activates the cyclosome/APC. Then,working in conjunction with a Ub-conjugating enzyme called E2-C in clams(an organism favored by cell-cycle researchers), the cyclosome marks themitotic cyclins for degradation by the proteasome (Aristarkhov et al.,1996, Proc. Nat. Acad. Sci. U.S.A., 93: 9303-9307); Ub-directeddestruction of the mitotic cyclins leads to the inactivation of Cdc2 andthe degradation of the ‘glue” proteins, so that sister chromatids areallowed to segregate into the two daughter cells. E2-C and its humanhomologue, the ubiquitin-conjugating human enzyme UbcH10, have beencharacterized in detail (Townsley et al., 1997, Proc. Natl. Acad. Sci.U.S.A., 94: 2362-2367).

ii Cell Signalling Pathways

Proteins that control cell-cycle progression may respond toenvironmental cues, such as are provided by growth factors. Growthfactor-stimulated signaling pathways are, themselves controlled in partby ubiquitination. One of the best studied examples is the NFκB pathway(see below). Binding of the cytokine tumor necrosis factor-α(TNF-α) tocell-surface receptors, or the occurrence of another proinflammatory orstress event (e.g. hypoxia), initiates a signaling cascade thatactivates NFκB (see below) and c-Jun, transcription factors that governthe proliferative response in cells.

Ubiquination may be involved in regulating the amount of a receptorpresent on the cell membrane. Stimulation of the Met tyrosine-kinasereceptor by the ligand hepato-cyte growth factor/scatter factor (HGF/SFDspurs the embryonic development of a variety of mammalian tissues,including liver, placenta, and muscles). For example, it has beenreported that HGF/SF stimulates the degradation of the Mettyrosine-kinase receptor by proteasomes in a human sarcoma cell line(Jeffers et al., 1997, Mol. Cell. Biol., 17: 799-808). In the absence ofHGF/SF, this receptor is cleaved by an unknown protease and the fragmentcontaining the tyrosine-kinase activity remains embedded in the cellmembrane. According to Hopkin et al. (1997, supra), it has beenpostulated that the presence of an unregulated tyrosine kinase in themembrane could be dangerous and that Ub-targeted degradation is intendedto rid the cell of the membrane-embedded kinase fragment before damagecan occur.

It is thought that the proteasome will cleave any ubiquitinated proteinwith which it comes in contact; however, different receptors mayrecognize substrates bearing Ub chains that differ in internal. The2-megadalton proteasome complex, which comprises four stacked rings of αand β protein subunits with a series of protease-active sites lining theinside of the resulting tube, recognizes a subset of ubiquitin chainsvia the S5 protein subunit. After a Ub-tagged protein binds to theproteasome complex, it is unfolded in order to facilitate passagethrough the proteasome pore into the proteolytic chamber. Mutationalinactivation of the S5 proteasome subunit results in a specific subsetof ubiquitinated proteins being spared from degradation (van Nocker etal., 1996, Mol. Cell. Biol., 16: 6020-6028). It is this selectivitywhich suggests that the proteasome may possess more than one receptorfor detecting Ub-conjugated proteins.

NFκB has been implicated in the etiology of immune disorders. Adams etal. (WO 96/13266) teach inhibition of proteasome activity, whichmediates the activation of NFκB, to treat autoimmune diseases.

Similarly, Brand et al. (WO 95/24914) teach that new, as well asexisting, proteasome inhibitors may be used to treat autoimmunediseases.

Further, according to Palombella et al. (WO 95/25533; page 7, lines16-23), Goldberg et al. are said to teach methods and drugs that inhibitantigen processing for the treatment of autoimmune diseases.

According to Kopp and Ghosh (1994, Science, 265: 956-969) and Grilli etal. (1996, Science, 274: 1383-1385), salicylate and glucocorticoids,anti-inflammatory drugs that are inhibitors of NFκB, are widely used totreat established cases of autoimmune diseases.

In addition, NFκB is said to said to be a positive transcriptionalregulator of inducible nitric oxide synthase (iNOS), which in turnmediates cytokine-induced inhibition of insulin secretion by pancreaticcells of the islets of Langerhans (Kwon et al., 1995, Endocrinonlogy,136: 4790-4795); inhibition of NFκB activity suppresses this phenotype.

There is need in the art for improved methods of treating autoimmunedisorders.

SUMMARY OF THE INVENTION

The invention provides a method of detecting autoimmune disease in amammal, comprising providing a biological sample from a mammal anddetecting proteasome activity, wherein a reduction in proteasomeactivity from a basal state is indicative of autoimmune disease.

As used herein, the term “autoimmune disease” refers to a disorderwherein the immune system of a mammal mounts a humoral or cellularimmune response to the mammal's own tissue or has intrinsicabnormalities in its tissues preventing proper cell survival withoutinflammation.

Examples of autoimmune diseases include, but are not limited to,diabetes, rheumatoid arthritis, multiple sclerosis, lupus erythematosis,myasthenia gravis, sclerodenna, Crohn's disease, ulcerative colitis,Hashimoto's disease, Graves' disease, Sjögren's syndrome, polyendocrinefailure, vitiligo, peripheral neuropathy, graft-versus-host disease,autoimmune polyglandular syndrome type I, acute glomerulonephritis,Addison's disease, adult-onset idiopathic hypoparathyroidism (AOIH),alopecia totalis, amyotrophic lateral sclerosis, ankylosing spondylitis,autoimmune aplastic anemia, autoimmune hemolytic anemia, Behcet'sdisease, Celiac disease, chronic active hepatitis, CREST syndrome,dermatomyositis, dilated cardiomyopathy, eosinophilia-myalgia syndrome,epidermolisis bullosa acquisita (EBA), giant cell arteritis,Goodpasture's syndrome, Guillain-Barré syndrome, hemochromatosis,Henoch-Schönlein purpura, idiopathic IgA nephropathy, insulin-dependentdiabetes mellitus (IDDM), juvenile rheumatoid arthritis, Lambert-Eatonsyndrome, linear IgA dermatosis, myocarditis, narcolepsy, necrotizingvasculitis, neonatal lupus syndrome (NLE), nephrotic syndrome,pemphigoid, pemphigus, polymyositis, primary sclerosing cholangitis,psoriasis, rapidly-progressive glomerulonephritis (RPGN), Reiter'ssyndrome, stiff-man syndrome and thyroiditis.

As used herein, the term “diabetes” refers both to the type I form ofthe disease and to type II cases that share only an islet cell defectwith type I.

Symptoms common to many types of autoimmune dysfunction include, but arenot limited to: fatigue; inflammation; paresis; joint stiffness, pain orswelling; skin lesions or nodules; skin discoloration; enzymaticimbalances; tissue degeneration. Examples of such symptoms as pertain tospecific autoimmune diseases are described hereinbelow in theDescription section. Such symptoms or, alternatively, measurements oftissue death/destruction, may be used either as diagnostic indicators ofthe presence of an autoimmune disease, or as indices by which to assessthe efficacy of treatment thereof.

In the treatment of autoimmune disease, a therapeutically effectivedosage regimen should be used. By “therapeutically effective”, onerefers to a treatment regimen sufficient to restore the the mammal tothe basal state, as defined herein, at the cellular or tissue site ofmanifestation or to prevent an autoimmune disease in an individual atrisk thereof or restore the mammal's immune system to the basal state.Alternatively, a “therapeutically effective regimen” may be sufficientto arrest or otherwise ameliorate symptoms of an autoimmune disease.Generally, in the treatment of autoimmune diseases, an effective dosageregimen requires providing the medication over a period of time toachieve noticeable therapeutic effects; such a period of time may beginat, or even before, birth and continue throughout the life of theindividual being treated. Methods of treatment are discussed in detailin the Description section, below.

As used herein, the term “biological sample” refers to a whole organismor a subset of its tissues, cells or component parts (e.g. body fluids,including but not limited to blood, mucus, lymphatic fluid, synovialfluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood,urine, vaginal fluid and semen). “Biological sample” further refers to ahomogenate, lysate or extract prepared from a whole organism or a subsetof its tissues, cells or component parts, or a fraction or portionthereof Lastly, “biological sample” refers to a medium, such as anutrient broth or gel in which an organism has been propagated, whichcontains cellular components, such as proteins or nucleic acidmolecules.

As used herein, the term “organism” refers to all cellular life-forms,such as prokaryotes and eukaryotes, as well as non-cellular, nucleicacid-containing entities, such as bacteriophage and viruses.

As used herein, the term “mammal” refers to a member of the classMammalia, including a human.

It is contemplated that procedures useful for the detection of proteinsor nucleic acids and biological activities thereof include, but are notlimited to, immunological assays, such as immunoblotting,immocytochemistry, immunohistochemistry or antibody-affinitychromatography, electrophoretic analysis, such as one- ortwo-dimensional SDS-PAGE, Northern or Southern analysis, in vivo or invitro enzymatic activity assay, the polymerase chain reaction (PCR),reverse-transcription PCR (RT-PCR), in situ nucleic acid hybridization,electrophoretic mobility shift analysis (EMSA), transcription assay, orvariations or combinations of these or other techniques such as areknown in the art.

As used herein, the term “proteasome” refers to a multi-subunit proteincomplex in the cytoplasm of eukaryotic cells which recognizes andselectively cleaves ubiquitinated protein molecules to mediate eitheractivation or degradation of the protein so recognized and cleaved.

As used herein in reference to proteasome activity, the term “reduction”refers to the failure of the proteasome to cleave a target ubiquitinatedprotein at as few as one-, more than one-, or even as many as all of thesites that it normally (ie., in a genetically wild-type or otherwisehealthy individual) recognizes and cleaves in that protein. Preferably,such a reduction involves failure to cleave the target protein at 5-10%of sites, more preferably, at 20-50% of sites, and most preferably at75-100% of such sites. Different numbers and/or patterns of sites ondifferent proteins are cleaved by the proteasome. The term “differentproteins” refers to protein molecules that differ in amino acid sequencein at least one position. Promiscuous cleavage (i.e., at a site notnormally recognized and cleaved) of a protein by the proteasome isdefined as a reduction only if such aberrant cleavage is accompanied bythe failure of the proteasome to cleave a site normally recognized andcleaved.

As used herein, the term “basal state” refers to the level of activityof a protein, nucleic acid or other molecule where autoimmune disease isnot present, i.e. a “normal level” of activity. The basal state isobserved in genetically wild-type or otherwise healthy individuals, aswell as in individuals who have a propensity for an autoimmune disease(as judged by genetic or environmental criteria known to those of skillin the medical art) but have not yet developed such a disease and evenindividuals who are in the early stages of an autoimmune disease buthave not, for example, become actively symptomatic.

Preferably, the biological sample comprises protein.

It is contemplated that the protein of a biological sample of use in theinvention may be crude (i.e., in an unfractionated cell lysate),partially-purified or isolated, and either naturally-occurring orproduced by recombinant techniques, such as the expression of a cDNA orother gene sequence cloned from a mammal.

In a preferred embodiment, a reduction in proteasome activity isdetected.

A reduction in proteasome activity may be observed as a reduction in theactivation of transcription factors (among them, NFκB) as judged eitherby observation of the physical properties of such a protein (forexample, antigenicity or molecular weight, as judged by sedimentation orelectrophoretic mobility) that are characteristic of its pre-activationform or by the absence of mRNA (or the protein encoded by such amessage) resulting from the transcription of a gene that is positivelyregulated by the protein in a biological sample. In addition, areduction in the proteolytic processing of a protein normally cleaved bythe proteasome (such as an MHC antigen, which is cleaved by theproteasome prior to transport to- and presentation on the cell surface).

Preferably, the reduction in proteasome activity comprises a reductionof proteolytic processing of NFκB, p105, p100, IκB, or a subunit thereof

Methods for the detection of a reduction in proteolytic processing ofNFκB are as described in detail hereinbelow in Example 2.

Preferably, the mammal is a human.

It is preferred that the autoimmune disease is an HLA class II-linkeddisease.

As used herein, the term “HLA class II-disease” refers to thoseautoimune diseases showing statistical risk factors for diseasepenetrance attributed to HLA class II genes or to neighboring genes.

The term “HLA” (for “human lymphocyte antigen”) refers to genes of thehuman major histocompatibility complex (MHC) or their protein products.In mice, the genetic region corresponding to or homologous with the HLAis termed the H2 complex.

In another preferred embodiment, the autoimmune disease is selected fromthe group that includes those diseases listed above as autoimmunediseases.

Another aspect of the present invention is a method of detectingautoimmune disease in a mammal, comprising providing a biological samplefrom a mammal and detecting protein ubiquitination, wherein a reductionin protein ubiquitination from a basal state is indicative of autoimmunedisease.

As used herein in reference to protein ubiquitination, the term“reduction” refers to the failure of ubiquitinating enzymes toubiquitinate a target protein at as few as one-, more than one-, or evenas many as all of the sites that they normally (ie., in a geneticallywild-type or otherwise healthy individual) recognize and ubiquitinate inthat protein. Preferably, such a reduction involves failure toubiquitinate the target protein at 10-20% of sites, more preferably, at40-50% of sites, and most preferably at 80-100% of sites. Differentnumbers and/or patterns of sites on different proteins are ubiquitinatedby the ubiquitinating enzymes. The term “different proteins” refers toprotein molecules that differ in amino acid sequence in at least oneposition. Promiscuous ubiquitination (i.e., at a site not normallyrecognized and ubiquitinated) of a protein by the ubiquitinating enzymesis defined as a reduction only if such aberrant ubiquitination isaccompanied by the failure of the ubiquitinating enzymes to ubiquitinatea site normally recognized and ubiquitinated.

It is preferred that the biological sample comprises protein.

It is additionally preferred that a reduction in protein ubiquitinationis detected for a protein.

Preferably, the mammal is a human.

It is preferred that the autoimmune disease is an HLA class II-linkeddisease.

In another preferred embodiment, the autoimmune disease is selected fromthe group that includes those diseases listed above.

The invention also encompasses a method of detecting autoimmune diseasein a mammal, comprising providing a biological sample from a mammal anddetecting protein phosphorylation, wherein a reduction in proteinphosphorylation from a basal state is indicative of autoimmune disease.

As used herein in reference to protein phosphorylation, the term“reduction” refers to the failure of a protein kinase to phosphorylate atarget protein at as few as one-, more than one-, or even as many as allof the sites that it normally (ie., in a genetically wild-type orotherwise healthy individual) recognizes and phosphorylates in thatprotein. Preferably, such a reduction involves failure to phosphorylatethe target protein at 2-10% of sites, more preferably, at 25-50% ofsites, and most preferably at 90-100% of sites. Different numbers and/orpatterns of sites on different proteins are phosphorylated by proteinkinases. The term “different proteins” refers to protein molecules thatdiffer in amino acid sequence in at least one position. Promiscuousphosphorylation (i.e., at a site not normally recognized andphosphorylated) of a protein by a protein kinase is defined as areduction only if such aberrant phosphorylation is accompanied by thefailure of the protein kinase to phosphorylate a site normallyrecognized and phosphorylated.

It is preferred that the biological sample comprises protein.

It is also preferred that a reduction in protein phosphorylation isdetected.

Preferably, the mammal is a human.

It is preferred that the autoimmune disease is an HLA class II-linkeddisease.

In another preferred embodiment, the autoimmune disease is selected fromthe group provided above.

Another aspect of the present invention is a method of detectingautoimmune disease in a mammal, comprising providing a biological samplefrom a mammal and detecting NFκB activity, wherein a reduction in NFκBactivity from a basal state is indicative of autoimmune disease.

As defined herein with regard to NFκB activity, the term “reduction”refers to a loss of the ability of NFκB to direct the transcription ofgenes whose cis-regulatory sequences comprise an NFκB recognition site,wherein such a site is normally bound and transcription of the geneactivated by NFκB. Preferably, such a reduction is in the range of 5-10%of the basal state level of activity, more preferably 25-50% and mostpreferably 70-100%.

Preferably, the biological sample comprises protein.

It is preferred that the biological sample comprises a nucleic acid.

As used herein, the term “nucleic acid” refers to a DNA molecule, suchas genomic DNA or cDNA, and also to RNA. A nucleic acid may be double-or single-stranded, circular or linear and may be naturally-occurring,recombinant or synthetic (produced by either enzymatic or chemical meansas a known in the art); if recombinant or synthetic, a nucleic acidmolecule may comprise sequences which are known to occur naturally orwhich are novel.

It is preferred that a reduction in said NFκB activity is detected.

As stated above, a reduction in in NFκB activity may be determinedeither through its failure to direct the transcription of downstreamgenes, physical characteristics or DNA- or protein-binding activity incomparison to those of the basal state. NFκB activity may be assayedeither in vivo or in vitro using an NFκB-dependent reporter geneexpression construct and a substrate for enzymatic detection (such aschloramphenicol acetyl transferase or β-galactosidase, depending on thespecificity of the enzyme encoded by the reporter gene), whereincomparative quantitation of the product of the diagnostic enzymaticreaction (or, in the absence of a reaction substrate, the level of thereporter mRNA or its encoded protein) in biological samples derived froma test subject and a normal control indivicual allow for the assessmentof NFκB functional loss. Alternatively, immunological or otherbiochemical determination of whether or not IκB has been cleaved fromNFκB may be made, as described above and in Example 2, below.

Preferably, the mammal is human.

It is preferred that the autoimmune disease is an HLA class II-linkeddisease.

In another preferred embodiment, the autoimmune disease is selected fromthe group that includes those diseases listed above.

The invention also provides a method of detecting autoimmune disease ina mammal, comprising providing a biological sample from a mammal anddetecting cell survival or growth, wherein cell death prior to directlymphocyte or antibody attack in a tissue that is a suspected target ofan autoimmune disease is indicative of the autoimmune disease.

As used herein, the term “growth” refers to mitosis or differentiation(acquisition of cell surface marders or specialized functions, e.g.protein production, indicative of a mature cell type.

As used herein, the tern “tissue” refers to intact tissue or tissuefragments, such that the cells are sufficiently aggregated (associated)so as to form a cohesive mass. A tissue may comprise an entire organ(e.g. the pancreas, the thyroid, a muscle, or others) or other system(e.g. the lymphatic system) or a subset of the cells thereof; therefore,a tissue may comprise 0.1-10%, 20-50% or 50-100% of the organ or system(e.g. as is true of islets of the pancreas).

Examples of tissue types that are the targets of autoimmune diseaseinclude, but are not limited to, blood, lymph, the central nervoussystem (including brain or spinal cord gray or white matter), liver,kidney, spleen, heart muscle or blood vessels, cartilage, ligaments,tendons, lung, pancreas (in particular, pancreatic islets ofLangerhans), lacrimal ducts, melanocytes, the adrenal cortex, skin, theintestinal tract (in particular, the luminal epithelium and the colon),ovary, testes, prostate, and regions such as joints, nerve/blood vesseljunctions, salivary glands, bones, specific tendons or ligaments.

As used herein, the term “cells” is defined as including dissociatedcells, intact tissue or tissue fragments.

As used herein, the term “suspected target” refers to a tissue that isdamaged in the course of an autoimmune disease of which a mammal isbelieved to suffer or to be at risk of suffering.

It is contemplated that an individual is at risk of an autoimmunedisease based either upon family history, the results of genetictesting, exposure (either after birth or in utero) to a substance suchas is known to trigger autoimmune disease (see, below, the descriptionof animal models of autoimmune disease); such an individual is“suspected of suffering” (see below) or “suspected of harboring” anautoimmune disease or is said to have a “propensity” for developing sucha disease.

Preferably, the sample is obtained from the mammal at an early stage inthe disease prior to or early in the formation of autoantibodies againstthe tissue.

As used herein, the term “prior” may refer to a period of timeimmediately before autoantibodies first are or would expected to beformed in an individual with a propensity for autoimmune disease.“Prior” may be used to indicate a time weeks, months or years before theappearance of autoantibodies. It is contemplated that in an individualsuspected of being at risk for an autoimmune disease, this may be asearly as birth or even during the prenatal period.

As used herein, the term “early” refers to a stage of an autoimmunedisease preceding complete target tissue destruction by the immunesystem.

Preferably, cell death is detected in a tissue that is a suspectedtarget of autoimmune disease prior to the formation of autoantibodies.

It is preferred that the biological sample comprises cells of a tissuethat is a suspected target of autoimmune disease.

It is additionally preferred that the mammal is a human.

Preferably, the autoimmune disease is an HLA class II-linked disease.

In another preferred embodiment, the autoimmune disease is selected fromthe group that includes those diseases listed above.

The invention also encompasses a method of treating an autoimmunedisease in a mammal, comprising administering to a mammal suspected ofsuffering from an autoimmune disease an agent which restores proteinubiquitinating enzyme function in an amount and for a time sufficient toresult in normal protein ubiquitination in the mammal.

As used herein, the term “agent” refers to a biochemical substanceselected from the group that includes, but is not limited to, proteins,peptides or amino acids; nucleic acids such as DNA, such as full-lengthgenes or fragments thereof derived from genomic, cDNA or artificialcoding sequences, gene regulatory elements, RNA, including mRNA, tRNA,ribosomal RNA, ribozymes and antisense RNA, oligonucleotides andoligoribonucleotides, deoxyribonucleotides and ribonucleotides;carbohydrates; lipids; proteoglycans; such agents may be administered asisolated (purified) compounds or in crude mixtures, such as in a tissue,cell or cell lysate. Alternatively, “agent” may refer to an organic orinorganic chemical as is known in the art.

Methods of administering a therapeutic agent include, but are notlimited to, topical application (e.g., for skin lesions), intravenousdrip or injection, subcutaneous, intramuscular, intraperitoneal,intracranial and spinal injection, ingestion via the oral route,inhalation, trans-epithelial diffusion (such as via a drug-impregnated,adhesive patch) or by the use of an implantable, time-release drugdelivery device, which may comprise a reservoir of exogenously-producedagent or may, instead, comprise cells that produce and secrete thetherapeutic agent.

As used herein, the term “ubiquitinating enzyme function” refers to thecovalent attachment of one or more ubiquitin molecules to a protein bymembers of the several classes of ubiquitinating enzymes, which includeubiquitin-activating enzymes (E1, which prime ubiquitin for attachmentto a protein), ubiquitin-conjugating enzymes (E2, which bind primedubiquitin for transfer to a target protein and ubiquitin ligases (E3,which catalyze the linkage of ubiquitin to specific sites on the targetprotein, which sites vary in number and type from protein to protein, asdiscussed above).

As used herein with regard to protein ubiquitination, the term “restore”refers to a return of the ubiquitination of at least one site which isnormally ubiquitinated (that is, a site that is ubiquitinated in thebasal state, as defined above) and, preferably all such sites, but isnot ubiquitinated in the course of an autoimmune disease. Preferably, inthe restoration of a normal level and pattern of ubiquitination, 50% ofsuch sites are restored, more preferably, 60-85% and, most preferably,90-100%. Such percentages include only the ubiquitination of sites thatare normally ubiquitinated in the protein in question. In addition, anelevation of ubiquitination beyond 100% of normal values is notencompassed by this definition. It is contemplated that a restoration issufficient to allow proper (i.e., that which is qualitatively comparableto that observed in the basal state) recognition and cleavage of theprotein so ubiquitinated by the proteasome.

Preferably, the agent is selected from the group that consists of aprotein and a nucleic acid that encodes that protein.

It is preferred that the protein is selected from the group thatincludes a ubiquitin-activating enzyme (E1), a ubiquitin-conjugatingenzyme (E2) and ubiquitin-ligases (E3).

Examples of human homologues of the yeast ubiquitination enzymesinclude, but are not limited to UbcH5 (which functions as an E2) and theMDM2 oncoprotein, which acts as a ubiquitin ligase, or E3.

Preferably, the agent is a nucleic acid which encodes an antisense RNAor a ribozyme.

It is preferred that the mammal is a human.

It is additionally preferred that the autoimmune disease is an HLA class11-linked disease.

In another preferred embodiment, the autoimmune disease is selected fromthe group that includes those diseases listed above.

Another aspect of the present invention is a method of treating anautoimmune disease in a mammal, comprising administering to a mammalsuspected of suffering from an autoimmune disease an agent whichrestores NFκB activity in an amount and for a time sufficient to resultin normal NFκB activity in the mammal.

As used herein, the term “normal NFκB activity” refers to a value thatis at least 25% of the activity of one or more of NFκB and its subunitsp50, p105 and p65 observed in the basal state, as defined herein above,preferably in the range of 30-90% and most preferably in the range of95-100%. “Normal NFκB activity” may not exceed 100% of NFκB basal stateactivity.

Preferably, the agent is selected from the group that consists of aprotein and a nucleic acid that encodes that protein.

It is preferred that the protein is selected from the group thatincludes a mutant- or wild-type NFκB p50, NFκB p52, a competitor of IκBthat does not bind NFκB p50 or NFκB p65 (e.g., the IκB mutant describedin Ex. Jour. Biol. Chem., 1998, 273:2931, herein incorporated byreference), a mutant- or wild-type NFκB p65, tumor necrosis factor-α,E-selectin, I-cam, and V-cam, interleukin-2, interleukin-6, granulocytecolony-stimulating factor, interferon-β, Lmp2, Lmp7, aubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), aubiquitin-ligase (E3), a ubiquitin deconjugating enzyme (UCH), a proteinkinase, a proteasome subunit and an antibody directed against one of the240 kD and 200 kD human erythrocyte proteasome inhibitors, OF-2 and IκB.

In another preferred embodiment, the agent is selected from the groupthat consists of a ribozyme, an antisense RNA molecule, a DNA moleculethat encodes a said ribozyme, and a DNA molecule that encodes a saidantisense RNA molecule.

Preferably, the ribozyme or antisense RNA molecule is directed againstone of the 240 kD and 200 kD human erythrocyte proteasome inhibitors,OF-2 and IκB.

It is preferred that the mammal is a human.

It is additionally preferred that the autoimmune disease is an HLA classII-linked disease.

In another preferred embodiment, the autoimmune disease is selected fromthe group that includes those diseases listed above.

Another aspect of the present invention is a method of treating anautoimmune disease in a mammal, comprising administering to a mammalsuspected of suffering from an autoimmune disease resulting from areduction in the activity of NFκB, DNA repair factor TFIIH, STATtranscription factor, ubiquitination, phosphorylation or the proteasomean agent which restores lymphocyte maturation in an amount and for atime sufficient to result in normal lymphocyte maturation in the mammal.

It is preferred that the agent is selected from the group that consistsof a protein and a nucleic acid that encodes that protein.

It is additionally preferred that the protein is selected from the groupthat includes apolipoprotein B100, DNA repair factor TFIIH, STATtranscription factors a mutant- or wild-type NFκB p50, a mutant- orwild-type NFκB p65, tumor necrosis factor-α, E-selectin, I-cam, andV-cam, interleukin-2, interleukin-6, a ubiquitin deconjugating enzyme(UCH), colony-stimulating factor, interferon-β, Lmp2, Lmp7, aubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (2), aubiquitin-ligase (E), a protein kinase, a proteasome subunit and anantibody directed against one of the 240 kD) and 200 kD humanerythrocyte proteasome inhibitors, OF-2 and IκB.

Preferably, the agent is selected from the group that includes aribozyme, an antisense RNA molecule, a DNA molecule that encodes aribozyme and a DNA molecule that encodes an antisense RNA molecule.

It is preferred that the ribozyme or antisense RNA molecule is directedagainst one of the 240 kD and 200 kD human erythrocyte proteasomeinhibitors, OF-2 and IκB.

It is additionally preferred that the mammal is a human.

Preferably, the autoimmune disease is an HLA class II-linked disease,

In another preferred embodiment, the autoimmune disease is selected fromthe group that includes those diseases listed above.

A final aspect of the present invention is a method of treating anautoimmune disease in a mammal, comprising administering to a mammalsuspected of suffering from an autoimmune disease resulting from areduction in the activity of NFκB, DNA repair factor TFIIH, STATtranscription factor, or the proteasome an agent which restores the cellcycle in an amount and for a time sufficient to result in normalsurvival of cells of a tissue that is susceptible to an autoimmunedisease prior to the formation of autoantibodies, prior to cell death orprior to cellular attack against the cells in the

As defined herein, “normal survival of cells” is at least a 10% cellsurvival rate relative to that observed in the basal state. Preferably,“normal survival of cells” is in the range of 25-50% or even 75-100%;however, “normal survival of cells” does not encompass cell survival ata rate higher than 100% of that observed in the basal state. In otherwords, “normal survival of cells” does not refer to hyperproliferationof cells.

Preferably, the agent is selected from the group that includes a proteinand a nucleic acid that encodes that protein.

It is preferred that the protein is selected from the group thatincludes a cyclin, a cyclin-dependent kinase, apolipoprotein B100, DNArepair factor TFIIH, STAT transcription factor, a mutant- or wild-typeNFκB p50, a mutant- or wild-type NFκB p65, tumor necrosis factor-α,E-selectin, I-cam, and V-cam, interleukin-2, interleukin-6, granulocytecolony-stimulating factor, interferon-β, Lmp2, Lmp7, aubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), aubiquitin-ligase (E3), a ubiquitin deconjugating enzyme (UCH),a proteinkinase, a proteasome subunit and an antibody directed against one of the240 kD and 200 kD human erythrocyte proteasome inhibitors, OF-2 and IκB.

It is additionally preferred that the agent is selected from the groupthat includes a ribozyme, an antisense RNA molecule, a DNA molecule thatencodes a ribozyme and a DNA molecule that encodes an antisense RNAmolecule.

Preferably, the ribozyme or antisense RNA molecule is directed againstone of the 240 kD and 200 kD human erythrocyte proteasome inhibitors,OF-2 and IκB.

It is preferred that the mammal is a human.

It is additionally preferred that the autoimmune disease is an HLA classII-linked disease.

In another preferred embodiment, the autoimmune disease is selected fromthe group that includes those diseases listed above.

A final aspect of the invention is a method for screening for amodulator of LMP2 function, comprising the steps of contacting an assaysystem with a candidate modulator of LMP2, wherein in the system,proteasome-mediated cleavage of a ubiquitinated protein occurs, andmonitoring cleavage of the ubiquitinated protein, wherein a change incleavage resulting from the contacting indicates that the candidatemodulator is effective as a modulator of LMP2 function.

Further features and advantages of the invention will become more fullyapparent in the following description of the embodiments and drawingsthereof, and from the claims.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 shows the association of NFκBp65 with a cellular serine kinase.FIG. 1A: GST-NFκBp65 and GST-CTD were expressed in BL21pLysS E. Colicells and purified by selective absorption to glutathionsepharose beads.GST-NF-κBp65 was incubated with cytosolic and nuclear extracts. Reactionmixtures were washed in PBS. The precipitated complexes were thenincubated with GST-CTD of RNA polymerase II large subunit under thekinase buffer containing γ-[³²P]ATP as previously described (Hayashi etal., 1993, J. Biol. Chem., 268: 26790-26795; Faustman et al., 1989,Diabetes, 38: 1462-1468). The phosphorylated products were separated on12% SDS-PAGE and visualized by autoradiography. One-fortieth of theinput (I) and supernatant (S) fractions and {fraction (1/40)} of thelast wash (W) and pellet (P) fractions were used for in vitro kinasereaction. FIG. 1B: In vitro kinase reactions were performed under thedifferent conditions containing the individual indicated amount of theprecipitated complexes, GST-CTD. FIG. 1C: Selective inhibition ofphosphorylation activity by DRB. In vitro kinase assays were carried outand phosphorylated products were separated by SDS-PAGE and visualized byautoradiography (upper panel). Quantitation of phosphorylated GST-CTDwas performed with BAS 3000 phosphoimager and were plotted out (lowerpanel). The indicated concentration of DRB were included in each kinasereaction mixture. FIG. 1D: Phosphoamino acid analysis of in vitro-labeled GST-CTD. GST-CTD of RNA polymerase II large subunit werephosphorylated in the in vitro kinase reaction and resolved by SDS-PAGE.The phosphorylated form of GST-CTD was excised from the gel andprocessed for phosphoamino acid analysis. The phosphoamino acids wereseparated by electrophoresis by standard methods, and the migration ofthe phosphoamino acid standards were visualized by ninhydrin staining,as indicated. FIG. 1E: Transactivation domain of NFκBp65 is sufficientfor the association with a cellular serine kinase. Cytosolic extracts ornuclear extracts were incubated with either GST, GST-NFκBp65Q417 orGST-NFκBp65C418. Precipitated complexes were incubated with GST-CTD ofRNA polymerase II large subunit in kinase buffer containing γ-[³²P]ATP.The phosphorylated products were separated on 12% SDS-PAGE andvisualized by autoradiography. One-fortieth of the input (1) andsupernatant (S) fractions and {fraction (1/40)} of the last wash (W) andpellet (P) fractions were used for the in vitro kinase reaction.

FIG. 2 presents the detection of ATP-binding proteins that associatewith NFκBp65, such as cellular serine kinases, by in vitro affinitylabeling. GST-NF-κBp65 (FIG. 2A) or GST-NF-κBp65C418 (FIG. 2B) wasincubated with cytosolic extract (left panel), nuclear extract (rightpanel). The precipitated complexes were then incubated with GST-CTDunder the kinase buffer containing γ-[³²P]ATP for in vitro kinase assay.The precipitated complexes were incubated with 8-azide-α-³²P) ATP inkinase buffer for the ATP-binding assay. The samples were irradiated bya UV lamp. The phosphorylated products or ATP affinity-labeled productswere separated on 12% SDS-PAGE and visualized by autoradiography. Onefortieth of the input (1) and supernatant (S) factions and {fraction(1/40)} of the last wash (W) and pellet (P) fractions were used for thein vitro kinase reaction. FIG. 1C: The cytosolic extract (left panel),nuclear extract (right panel) were incubated with anti-NFκBp65polyclonal antibody. Immunoprecipitation assays were performed and thenthe immunoprecipitated complexes were incubated with GST-CTD in kinasebuffer containing γ-[³²P]ATP for the in vitro kinase assay. Theimmunoprecipitated complexes were incubated with 8-azide-(α-³²P) ATP inkinase buffer for the ATP-binding assay. The samples were irradiated bya UV lamp. The phosphorylated products or ATP affinity-labeled productswere separated on 12% SDS-PAGE and visualized by autoradiography.One-fortieth of the input (I) and supernatant (S) fractions and{fraction (1/40)} of the last wash (W) and pellet (P) factions were usedfor in vitro kinase reaction.

FIG. 3 shows the induction of kinase activity by HIV-1 trans-activatortranscription factor (Tat). FIG. 3A: GST-NFκBp65 was incubated withcytosolic and nuclear extracts. The precipitated complexes werepre-incubated with either wild-type GST-Tat, or either of the mutantsGST-Tat K41A and GST-Tat Cys22 at 4° C. for minutes. The amount ofGST-Tat added into the reaction mixtures is indicated in the figure. Thereaction mixtures were then incubated with GST-CTD in kinase buffercontaining γ-[³²P]ATP. The phosphorylated products were separated on 12%SDS-PAGE and visualized by autoradiography. FIG. 3B: Graphicrepresentation of quantitation of phosphorylated GST-CTD.

FIG. 4 shows the association of NFκBp65 with Cdks. Cytosolic extracts ornuclear extracts were incubated with either GST-NFκBp65 (FIG. 4A, FIG.4C) or GST-NFκBp65 C418 (FIG. 4B). FIGS. 4A and 4B: The proteincomplexes were precipitated using GST-sepharose beads after incubationand immunoblotting. FIG. 4C: The protein complexes were precipitatedusing anti-NFκBp65 polyclonal antibody after incubation andimmunoblotting blotting with appropriate antibodies. One-fortieth of theinput (1) and supernatant (S) fractions and {fraction (1/40)} of thelast wash (W) and pellet (P) fractions were used for the immunoblottingblotting assay.

FIG. 5 shows the absence of association of NFκBp65 with Cdks in NODmice.

FIG. 6 shows DNA-binding activities of NFκB and other transcriptionfactors in lung tissue of BALB/C and NOD mice.

FIG. 7 presents the identification of NFκB DNA-binding protein inDNA/protein complexes using super-shift assay.

FIG. 8 presents κB sequence-binding activities in spleen cells fromBALB/C and NOD mice.

FIG. 9 shows the identification of κB sequence-binding protein inDNA/protein complexes using the super-shift assay.

FIG. 10 presents immunoblot analysis of the basal expression of NF-κBsubunits, IκBα and cyclin-dependent kinases in spleen cell cytosolic andnuclear extracts of male and female BALB/c and NOD mice.

FIG. 11 presents DNA-binding activity of NF-κB in Lmp-deficient T2cells.

FIG. 12 presents in vitro analysis of phosphorylation, ubiquitination,and proteolysis of p105 in cytosolic extracts of BALB/c and NOD mice andimmunoblot analysis of MHC-linked proteasome subunits.

FIG. 13 presents TNF-α cytotoxicity of spleen cells and embryonicfibroblasts derived from BALB/c and NOD mice.

FIG. 14 shows that NOD spleen cells, but not embryonic fibroblasts(MEF), lack expression of the MHC-encoded LMP2 proteasome protein andthe p50 subunit of NF-κB.

FIG. 15 presents blocking early p105 processing to the p50 subunit inNOD mouse spleen cells.

FIG. 16 presents a κB binding protein competition assay usingoligonucleotides comprising palindromic κB binding motif in BALB/c andNOD lymphocyte extracts.

DESCRIPTION OF THE INVENTION

The present invention is predicated on the discovery that NOD mice aredeficient for NFκB activity. As described herein below, the methods andof the present invention comprise restoration of proteasome function, orsimply that of NFκB, in the treatment of autoimmune disorders. Theinventive methods are therefore contrary to prior art methods and,indeed, unexpected, based upon prior art references, which teachsuppression of NFκB or of proteasome activity (and, consequently, thatof NFκB) as a method of treating autoimmune disorders (see above).Restoration of proteasome function or of NFκB activity may be directedat the proteasome, the ubiquitinating machinery or protein kinases.Alternatively, therapy may involve providing functional (active forms of) NFκB that is independent of the proteasome for activation or even theproducts of downstream genes normally under the transcriptional controlof NFκB or providing the cell with cytoplasmic forms of NFκB for cellcycle control and cell differentiation/viability. The object of suchtreatment is to inhibit the progression of an autoimmune disease or toprevent its clinical initiation, where “clinical initiation” refers tothe presentation of symptoms and to organ destruction.

Detection Defect in Proteolytic Processing

The invention contemplates detection of autoimmune disease by detectinga defect in proteasome activity. Such defects may be detected using thefollowing assays.

i The Proteasome

Proteasome activity may be assayed as previously described (Gaczynska etal., 1994, Proc. Natl. Acad. Sci. U.S.A., 91: 9213-9217, incorporatedherein by reference). Briefly, cells (whether cultured cells, or thoseof an model animal, such as a mouse) in which the efficacy of astimulator of proteasome activity is to be assayed prior toadministration to a human are homogenized in a Dounce homogenizer orother grinding device (e.g. a mortar and pestle or a blender) and thenby vortex mixing with glass beads in a homogenization buffer (40 mMTris.HCl, 5 mM MgCl₂, 2 mM ATP, 250 mM sucrose, pH 7.4). Fractionscontaining total 20S and 26 S proteasomes are isolated by differentialcentrifugation of homogenates: for 20 minutes at 10,000×g, then for 1hour at 100,000×g or for hours at 100,000×g. Pellets are solubilized in50 mM Tris.HCl, 5 mM MgCl₂, 2 mM ATP, 20% (volume/volume) glycerol, pH7.4. Resulting “proteasome fractions” are used for peptidase assays andWestern blot analysis. Degradation of the fluorogenic peptides,N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-MCA),N-tert-butoxycarbonyl-Leu Arg-Arg-7-amido-4-methylcoumarin (Boc-LRR-MCA)and N-carbobenzoyx-Leu-Leu-Glu-β-naphthylamide (Cbz-LLE-βNA) is assayedat 37° C., for 40 minutes or 1 hour in the presence of apyrase (5units/ml), as described previously described (Gaczynska et al., 1993,Nature, 365: 552-554, also incorporated herein by reference).

ii. Ubquitination

The invention contemplates detection of autoimmune disease by detectinga defect in the activity of ubiquitinating enzymes. Such defects may bedetected using the following assays; Western analysis with antibodiesdirected against active forms of ubiquitinating enzymes, observation ofeletrophoretic mobility of on a Western blot of the ubiquitinated formof a test protein or peptide relative to its non-ubiquitinated form orits proteolytically processed form relative to its unprocessed form incytoplasmic extracts of unknown ubiquitinating capacity, Northernanalysis to detect loss of mRNAs whose transcription is dependent upon aprotein which required ubiquitination or enzymatic or other assay todetermine the function of a protein or peptide incubated in acytoplasmic extract of unknown ubiquitinating capacity, wherein theprotein or peptide requires ubiquitination in order to undergoproteolytic activation. In vitro ubiquitination assays are known in theart (see Chen et al., 1995, Genes Dev., 9: 1586-1597; Corsi et al.,1995, J. Biol. Chem., 270: 8928-8935; Corsi et al., 1997, J. Biol.Chem., 272: 2977-2983; Mori et al., 1997, Eur. J. Biochem., 247:1190-1196; Verma et al., 1997, Mol. Cell Biol., 8: 1427-1437; Kumar etal., 1997, J. Biol. Chem., 272: 13548-13554).

iii. NFκB

The invention contemplates detection of autoimmune disease by detectinga defect in the activation of NFκB. Such defects may be detected usingthe following assays.

The presence or absence of NFκB activity may be assayed by immunologicalanalysis of protein from cells or individuals using anti-NFκB antibodies(in which one would expect to observe a band the size of IκB-free NFκB).Such protein may be derived from a biological sample, including, but notlimited to, a tissue, cell, cell lysate or body fluid from anindividual. Northern analysis using labeled nucleic acid probes specificfor transcripts that may be produced by the downstream targets of NFκB(i.e., genes which are transcriptionally activated by that protein) maybe performed. Alternatively, nuclear protein extracts may be preparedfrom such cells and tested for the ability to activate transcription invitro of a marker gene which is operatively linked to an NFκB-induciblegene regulatory sequence. Assays may be directed at individual NFκBsubunits, such as p50 and p65, as in Examples 1 and 2 below, wherein thecytoplasmic and nuclear functions of these subunits are tested in normaland autoimmune mice. In addition to activity, their processing from alarger protein or release from inhibitory substances may be assessed bymolecular and biochemical methods known in the art (such as PAGE orWestern analysis, as described below).

While these approaches are technically feasible, they may not bemedically expedient or even safe, as they entail removal of treatedcells from the patient. It is recommended that immunological analysis beperformed on serum protein extracts, using antibodies which are directedagainst products of genes under the control of NFκB which are secretedproteins, by methods described below.

Restoration of Normal Proteolytic Processing

The invention contemplates methods of treating autoimmunity by restoringproteolytic processing, based upon the observation that NFκB activity isabsent in the NOD mouse model of autoimmune disease. Restoration ofproteolytic processing, such as would result in the restoration of NFκBactivity, may be directed at the proteasome, the ubiquitinatingmachinery or protein kinases.

A Therapeutic Targets Suppression of Proteasome Inhibitors

The invention contemplates methods of treating autoimmunity by restoringproteolytic processing by blocking the activity of inhibitors ofproteasome function or changing the specificity of a proteasome subunitto favor activation of the substrate(s) deficient in an autoimmunedisease, so that correct protein processing is restored.

Inhibition of proteasome activity blocks the production of activatedNFκB and other essential proteins, as described above; therefore, inorder to promote correct protein processing, it may be necessary toinactivate cellular inhibitors of the proteasome. Such endogenousinhibitors of proteasome activities have been isolated. These includethe 240 kD and the 200 kD inhibitors isolated from human erythrocytes(Murakami et al., 1986, Proc. Natl. Acad. Sci. U.S.A., 83: 7589-7592; Liet al., 1991, Biochemistry, 30: 9709-715) and purified OF-2 (Goldberg,1992, Eur. J. Biochem., 203: 9-23).

Proteasome processed proteins leading to activation include P100andP105. Proteasome processed proteins leading to degradation includeTFIIH, Stat proteins, Jak proteins (Jak2, Jak1), Shc, Sp1, CDC25B, Kip1,p27, Serotonin N-acetyl transferase, IkB, P53, Cyclins, c-Fos, c-Jun,presenilin 1 FosL, tyrosine aminotransferase, and ornithinedecarboxylase.

Endogenous proteasome inhibitors may be inactivated by methods known inthe art, which methods include the administration of antibodies whichbind them specifically, the use of antisense RNA or ribozymes directedagainst the mRNAs which encode them (see below). Antibodies againstnumerous proteins are now publicly available, both through commercialand non-profit suppliers (e.g. ATCC); however, antibodies of use in theinvention may, if necessary, be prepared as described below.

Restoration of Wild-type Proteasome Function

The invention contemplates methods of treating autoimmune disease bydirect stimulation of proteasome function, thereby restoring orpreserving correct proteolytic processing.

Japanese Patent No. JP8322576, which is herein incorporated in full byreference, discloses proteasome activator PA28β(see also Chu-ping etal., 1992, J. Biol. Chem., 267: 10515; Dubiel et al., 1992, J. Biol.Chem., 267: 22369); both cloning of a cDNA from bovine tissues (e.g.liver, heart and red blood cells) and a method for the production of therecombinant polypeptide encoded by the cloned nucleic acids aredescribed by these references. PA28 (or PA28β) has a subunit molecularweight of 28,000, as judged by denaturing gel electrophoresis and anative molecular weight of approximately 180,000 as determined by gelfiltration and density gradient centrifugation; therefore, it is thoughtto exist as a hexameric protein complex. Dubiel et al. (1992, supra)further describe the isolation of a human protein of M_(r) approximately200,000 that activates proteasomes; this complex is a hexamer comprisingsubunits that display M_(r) of approximately 29,000 and 31,000 ondanaturing electrophoretic gels. This activator complex lacks intrinsicpeptidase activity, but stimulates proteolysis of certain substratesabout 60-fold, although activated proteasomes are unable to degradeubiquitin-lysozyme conjugates, bovine serum albumin or lysozyme;activation involves reversible binding of the activator complex toproteasomes. WO 95/27058 discloses a human protein complex (M_(r)approximately 29,000) which is a γ-interferon-inducible activator ofproteasome function. The sequences encoding each of these polypeptidesare of use in gene therapy according to the invention, as describedbelow. Alternatively, the proteins themselves may be administered bymethods known in the art (see also below).

In addition to proteasome-stimulating proteins, wild-type proteasomesubunits or other associated proteins (e.g. Lmp2, Lmp7) may beadministered if inactivating mutations are found within the sequencesencoding them or in the regulatory elements controlling thetranscription or these genes. While there exist many targets for suchspecifically-directed treatment, it should be noted that the discoveryof one such mutant (that found in the shared Lmp2/Tap promoter) isdescribed herein above (Yan et al., 1997, supra).

Restoration of Correct Ubiquitination/Phosphorylation

The invention contemplates methods of treating an autoimmune disease byrestoring correct patterns of ubiquitination and/or phosphorylation.

If proteolytic failure has been traced to a deficiency in ubiquitinationor phosphorylation, the missing activity may be supplemented eitherthrough the administration of a wild-type protein whose absence orinactivation is responsible for the deficiency or through gene therapy,in which a gene encoding such a protein is administered under theinfluence of transcriptional control elements (e.g., its own wild-typeelement or another strong promoter, e.g. thymidine kinase, heat-shock orothers as are known in the art). Such proteins may includeubiquitinating proteins of the E1, E2 and E3 families as well as “glue”proteins (all as described above); alternatively, protein kinases (e.g.,cyclin-dependent kinases; see also above) or cyclins may beadministered.

Restoration of NFκB Function

The invention contemplates methods of treating autoimmune diseases byrestoring NFκB function, which, in turn, restores the transcription ofNFκB-dependent genes.

As is true of the proteasome and of the ubiquitination and proteinphosphorylation machinery described above, it is possible to administerto cells of an organism in which NFκB carries an inactivating mutation,either in coding or regulatory sequences, a wild-type sample of the NFκBprotein or one or more copies of the gene encoding it; however, a secondscenario may instead be envisioned.

In the case in which NFκB activity is reduced or absent due to an‘upstream’ defect (that is, one involving activation by the proteasome,instead of- or in addition to a mutation in the NFκB gene itself), it ispossible to circumvent the need for proteolytic activation of NFκB byintroducing a constitutively-active version of the protein, such as onein which the IκB recognition site has been mutated such that IκB can nolonger bind to- and inactivate NFκB. Binding of NFκB to IκB occursthrough ankyrin repeats (as reviewed by Siebenlist et al., 1994, Ann.Rev. Cell. Biol., 10: 405-455); it is contemplated that sequencesencoding these repeats be deleted or mutated in an NFκB subunit p100 orp105 gene expression construct such that binding to IκB is significantlyimpaired or is eliminated. As a transcription/signalling factor whichremains active when it is no longer required may have undesirableconsequences, particularly in the absence of proteolytic which wouldnormally inactivate it under such circumstances, administration of sucha protein in limited doses or of a gene encoding it under atightly-regulated (i.e. inducible, rather than constitutive, promoter)may be necessary. Alternatively, such a protein may be expressed at alltimes, provided that an inhibitor thereof is co-administered; such aninhibitor may be an antibody directed against the protein, or anantisense RNA or ribozyme directed against the message encoding it, asdescribed below.

Inactivation of IκB may also be performed by methods described below,such as by the use of antibodies directed against it or of antisense RNAor ribozymes directed against the mRNA transcript encoding it.Preferably, such inactivation is transient, as it would otherwise leadto constitutive activation of NFκB, which activation is not, itself,normal.

The invention contemplates treatment of autoimmune disease using methodsdirected at the potential therapeutic targets discussed above. In thesection following, methods by which such treatment may be carried outare presented.

B. Therapeutic Methods Autoimmune Disorders in Humans

In order to provide effective treatment according to methodscontemplated by the invention, it is first necessary to identify thoseindividuals in need of treatment.

Genetic linkage studies have confirmed the MHC to be an importantcontributor to human autoimmune diseases such as type I diabetes,rheumatoid arthritis, lupus erythematosus, Hashimoto's disease, andmultiple sclerosis (Bach et al., 1994, Endocr Rev., 15: 516; Cudworthand Woodrow, 1976, Br. Med. J., 2: 846; Festenstein et al., 1986,Nature, 322: 64; Nerup et al., 1977, HLA and Disease, Munksgaard,Copenhagen; Todd et al., 1987, Nature, 329: 599; Van Endert et al.,1994, Diabetes, 43: 110). Other autoimmune disorders include Graves'disease, ulcerative colitis, Crohn's disease, polyendocrine failure,Sjögren's syndrome and others as listed above in the Summary.

The present invention is of use in the treatment of HLA class II-linkedautoimmune diseases such as those listed above. Diagnostic symptoms orother indicators may be used either to assess a patient for the presenceof- or susceptibility to such a disorder; in addition, improvement(i.e., a change toward the basal state, as defined above) in one or moreof these indicators is indicative of the efficacy of a given method oftreatment for such a disease.

Examples of autoimmune disease-related symptoms for severalrepresentative diseases are as follows:

Addisons's Disease

Addison's disease is a disorder characterised by failure of the adrenalgland and is often an autoimmune disorder involving destruction of theadrenal cortex and the presence of adrenal autoantibodies in thepatient's serum. The adrenal cortex is responsible for producing severalsteroid hormones including cortisol, aldosterone and testosterone. Inautoimmune Addison's disease and other forms of the disease, levels ofthese hormones are reduced. This reduction in hormone levels isresponsible for the clinical symptoms of the disease which include lowblood pressure, muscle weakness, increased skin pigmentation andelectrolyte imbalance.

Autoantibodies to the adrenal cortex may be identified for diagnosis ofAddison's disease using the technique of complement fixation orimmunofluorescence (Anderson et al., 1957, Lancet, 1: 1123-1124;Blizzard and Kyle, 1963, J. Clin. Invest., 42: 1653-1660; Goudie et al.,1968, Clin. Exp. Immunol., 3: 119-131; Sotosiou et al., 1980, Clin. Exp.Immunol., 39: 97-111). Radioimmunoassay and ELISA techniques using crudeadrenal membrane preparations are also of use in the invention(Stechemesser et al., 1985, J. Immunol. Methods, 80: 67-76; Kosowicz etal., 1986, Clin. Exp. Immunol., 671-679).

U.S. Pat. No. 5,705,400 discloses methods for the detection of adrenalautoantigen. Such assays are useful for the diagnosis of latent oractual autoimmune Addison's disease. These methods are brieflysummarized as follows:

1. Assay Based on a Radioactive Label

Purified adrenal autoantigen is labeled with a radioactive label such as¹²⁵I using one of many well-known techniques. The labeled material isthen incubated (1 hour at room temperature) with a suitably diluted(e.g. 1:20 in phosphate buffered saline) serum sample. Adrenalautoantibodies present in the test sample bind to the ¹²⁵I-labeledadrenal autoantigen and the resulting complex is precipitated byaddition of antibodies to human immunoglobulins or a similar reagent(e.g. solid phase Protein A). The amount of ¹²⁵I-labelled antigen in theprecipitate is then determined. The amount of adrenal autoantibody inthe test serum sample is a function of the amount of radioactivityprecipitated. The amount of adrenal autoantibody can be expressed as theamount of radioactivity in the pellet or more usually by includingdilution of an adrenal autoantibody-positive reference serum in theassay. Note that such techniques using autoantigens such as have beenidentified in other diseases may be broadly applied to the detection ofautoantibodies.

2. Assay Based on an Enzyme Label

Purified adrenal autoantigen is coated onto plastic wells of ELISAplates either directly onto plain wells or indirectly. The indirectmethod may involve coating the wells first with a monoclonal orpolyclonal antibody to adrenal autoantigen (the antibody is selected soas not to bind to the same site as adrenal autoantibodies) followed byaddition of adrenal autoantigen. Several other indirect coating methodsare well known in the art. After coating with autoantigen, suitablydiluted (e.g., 1:20 in phosphate buffered saline) test sera are added tothe wells and incubated (1 hour at room temperature) to allow binding ofadrenal autoantibody to the antigen coated onto the wells. The wells arethen washed and a reagent such as antihuman IgG conjugated tohorseradish peroxide is added. After further incubation (e.g., 1 hour atroom temperature) and washing, an enzyme substrate such asorthophenylene diamine is added and the color generated measured bylight absorbance. The amount of adrenal autoantibody in the test sampleis a function of the final color intensity generated. Results areexpressed as light absorbance or, more usually, by including dilution ofan adrenal autoantibody positive reference serum in the assay.

Ulcerative Colitis and Crohn's Disease

A number of human diseases result in the subject having a diseased gutin which digestion or absorption is impaired. Examples of autoimmunediseases in humans include chronic ulcerative gut diseases (e.g.,ulcerative colitis) and inflammatory gut diseases such as colitis andCrohn's disease.

In addition to impaired digestion and inflammation and/or ulteration ofthe intestinal tract, symptoms include pain, bleeding, abnormal stoolproduction and weight loss. Such symptoms may be assessed either bypatient interview or through techniques such as endoscopy and otherimaging techniques such as heavy metal (e.g. barium enema followed byX-ray), and scanning using CAT, positron emission tomography (PET),(magnetic resonance imaging) MRI or histological analysis (biopsy).

Lupus Erythematosus

As described by U.S. Pat. Nos. 5,695,785 and 5,700,641, and brieflysummarized here, lupus erythematosus is an autoimmune disease which isnot specific to a particular organ. The common type of lupuserythematosus, Discoid Lupus Erythematosus (DLE), affects exposed areasof the skin. The more serious and fatal form of the disease, SystemicLupus Erythematosus (SLE), affects a large number of organs and has achronic course with acute episodes. The external manifestations of SLEare lesions on the facial skin. In most cases, other areas of skin andthe mucosa are affected. Also observed are nephritis, endocarditis,hemolytic anemia, leukopenia and involvement of the central nervoussystem.

Many immunological phenomena have been observed with SLE. For example,the formation of antibodies against certain endogenous antigens has beenseen. These antibodies are directed against, for example, the basementmembrane of the skin, and against lymphocytes, erythrocytes and nuclearantigens. Antibodies which are directed against double-stranded DNA(ds-DNA) form with the latter complexes. These antibodies, together withcomplement, are deposited on small blood vessels and frequently resultin vasculitis. These deposits are especially dangerous when they occurin the renal glomeruli because they result in glomerulonephritis andkidney failure. The incidence of clinically detectable kidneyinvolvement is reported in the literature to be between 50 and 80%.

Of the multitude of autoreactive antibodies that spontaneously ariseduring the disease, high levels of circulating autoantibodies to DNA arethe best evidence of the pathogenesis. In SLE, there is almostinvariable presence in the blood of antibodies directed against one ormore components of cell nuclei. Certain manifestations in SLE seem to beassociated with the presence of different antinuclear antibodies andgenetic markers, which have suggested that SLE may be a family ofdiseases (Mills, 1994, Medical Progress, 33: 1871-1879). Lupusnephritis, especially diffuse proliferative glomerulonephritis, has beenknown to be associated with circulating antibodies to double stranded(native) DNA (Casals et al., 1964, Arthritis Rheum., 7: 379-390; Tan etal., 1964, J. Clin. Invest., 82: 1288-1294). The detection ofantinuclear antibodies is a sensitive screening test for SLE.Antinuclear antibodies occur in more than 95% of patients (Hochberg,1990, Rheum. Dis. Clin. North Am., 16: 617-639). Such autoantibodies maybe detected using DNA or other cellular components (such as smallnuclear ribonucleoprotein complexes) by the methods described above.

Sjögren's Syndrome

Tear film dysfunctions are collectively diagnosed askeratoconjunctivitis sicca (KCS) or, simply, dry eye (Holly et al.,1987, Internat. Opthalmol. Clin., 27: 2-6; Whitcher, 1987, Internat.Opthalmol. Clin., 27: 7-24). Lacrimal gland abnormalties falling intothe category of aqueous tear deficiencies, which are most frequentlyresponsible for dry eye states, include autoimmune disease. By far, thegreatest single cause of KCS worldwide, excluding those countrieswherein trachoma remains epidemic, is Sjögren's syndrome (Whitcher,1987, supra). This syndrome which is the second most common autoimmunedisease (Tabbara, 1983, “Sjögren's Syndrome” in The conrnea. ScientificFoundations and Clinical Practice, Smolin and Thoft, eds., Little Brownand Co., Boston, Mass., pp. 309-314; Daniels, 1990, “Sjögren'sSyndrome—in a nut shell” in Sjögren's Syndrome Foundation Inc. Report,Port Washington, N.Y.). This disease occurs almost exclusively infemales and is characterized by an insidious and progressive lymphocyticinfiltration into the main and accessory lacrimal glands, an immunemediated extensive destruction of lacrimal acinar and ductal tissues andthe consequent development of persistent KCS (Tabbara, 1983, supra;Moutsopoulos and Talal, 1987, in Sjögren's Syndrome Clinical andImmunological Aspects, Talal et al., eds., Springer Verlag, Berlin, pp.258-265; Talal and Moutsopoulos, 1987, in Sjögren's Syndrome. Clinicaland Immunological Aspects, Talal et al., eds., Springer Verlag, Berlin,pp. 291-295; Kincaid, 1987, in Sjögren's Syndrome. Clinical andImmunological Aspects, Talal et al., eds., Springer Verlag, Berlin, pp.25-33). In primary Sjögren's syndrome, which afflicts about 50% of thepatient population, the disease is also associated with an immunologicaldisruption of the salivary gland and pronounced xerostomia In secondarySjögren's, the disorder is accompanied by another autoimmune disease,which is most often rheumatoid arthritis and, less frequently, systemiclupus.

Dryness of the eyes, infiltration of lymphocytes into the lacrymalglands and the presence of autoantibodies are diagnostic criteria forSjögren's disease that are of use in the invention. The restoration one,more than one or even all of these indices to the basal state isindicative of effective treatment.

Type I Diabetes

Insulin dependent diabetes mellitus (IDDM) (also known as type Idiabetes) primarily afflicts young people. Although insulin is availablefor treatment the several-fold increased morbidity and mortalityassociated with this disease require the development of early diagnosticand preventive methods, as well as methods for the restoration of normalinsulin secretion (e.g., with islet therapy or regeneration osendogenous islets by methods described in detail below). As described inU.S. Pat. No. 5,691,448 and summarized briefly herein, the disappearanceof pancreatic β-cells (which are the insulin-secreting cells of theislets of Langerhans) precedes the clinical onset of IDDM. Among themost thoroughly studied autoimmune abnormalities associated with thedisease is the high incidence of circulating β cell-specificautoantibodies years prior to frank hyperglycemia, the typical clinicaldiagnosis. Family studies have shown that the autoantibodies appearprior to overt IDDM by years, suggesting a long prodromal period ofhumoral autoimmunity before clinical symptoms emerge, and have alsodocumented a slow, progressive loss of insulin response to intravenousglucose in the years preceding diagnosis. The presence of βcell-specific autoantibodies in the prediabetic period allows fordiagnosis according to the invention prior to critical β-cell depletionand insulin dependency. It has been estimated that only 10% of the totalβ-cell mass remains at the time of clinical onset (i.e., presentation ofelevated blood glucose levels relative to those observed in unaffectedindividuals, who represent the basal state, as defined above).

The target of autoantibodies in pancreatic β-cells in IDDM wereoriginally identified as both insulin and a 64 kD autoantigen byimmunoprecipitation experiments using detergent lysates of human islets(Baekkeskov et al., 1982, Nature, 298: 167-169). Antibodies to the 64 kDautoantigen precede the clinical onset of IDDM and have been shown tohave an incidence of about 80% at clinical onset and during theprediabetic period (Baekkeskov et al., 1987, J. Clin. Invest., 79:926-934; Atkinson et al., 1990, Lancet 335: 1357-1360; and Christie etal., 1988, Diabetologia, 31: 597-602. Many other autoantibodies exist,most directed against intracellular proteins.

A therapeutic agent is administered to a patient suspected of suffering-or suffering from established diabetes in an amount suffcient to inhibitor prevent further β-cell destruction/death. For individuals at risk ofIDDM or stiff man syndrome, the pharmaceutical agent is administeredprophylactically in an amount sufficient to either prevent or inhibitdestruction and death of the 0-cell. According to the invention, atherapeutic agent is administered in an amount and for a time sufficientto prevent or inhibit β cell destruction; β cell survival, as judged byimmunological detection of insulin, the level of serum glucose levels orrestoration of vigorous insulin stimulation to glucose challenge(intravenous glucose tolerance test, or IVGTT; Joslin, 1985, DiabetesMellitus, 20th Edition, eds. Marble et al., Lea & Febiger, Philadelphia,Pa.), is indicative of effective treatment.

Multiple Sclerosis

The symntoms of multiple sclerosis, such as those described in Treatmentof Multiple Sclerosis: Trial Design, Results and Future Perspectives,eds. Rudick and Goodkin, Springer-Verlag, N.Y., 1992 (particularly thosesymptoms described on pages 48-52), incorporated by reference as iffully set forth herein.

These multiple sclerosis symptoms include perturbations of pyramidalfunctions, for example the developement of paraparesis, hemiparesis,monoparesis, quadriparesis and the developement of monoplegia,paraplegia, quadriplegia, and hemiplegia. The symptoms of multiplesclerosis also include perturbations in cerebellular functions. Theseperturbations include the developement of ataxia, including truncal andlimb ataxia. When we refer to “paralytic symptoms of multiple sclerosis”we are refering to these perturbations in pyramidal and cerebellarfuntions. The symptoms of multiple sclerosis also include changes inbrain stem funtions, including development of nystamus and extraocularweakness along with dysarthria. Further symptoms include loss of sensoryfunction including decrease in touch or position sense and loss ofsensation in limbs. Perturbations in bowel and bladder function,including hesitancy, urgency, retention of bowel or bladder orincontinence, can also occur. Visual funtions, such as the developmentof scotoma, are also affected by multiple sclerosis. Cerebral functiondegeneration, including a decrease in mentation and the developemnt ofdementia, is also a symptom.

Inflamed MS and EAE (see below) lesions, but not normal white matter,sometimes have infiltrating CD4 T cells that respond to self antigenspresented by MHC class II-linked molecules like human HLA-DR2 (MS) ormurine I-A^(M) (EAE). The infiltrating CD4 Tcells (Th1 cells) produceproinflammatory cytokines interleukin(IL)-2, interferon (IFN)-γ, andtumor necrosis factor (TNF)-α that activate antigen-presenting cellslike macrophage to produce inflammatory cytokines (IL-1β, IL-6, andIL-8) and IL-12. The I-12 induces further IFN-γ synthesis. The imbalanceof one or more of these proteins relative to other cellular factors maybe assayed by biochemical or immunological methods as are known in theart. Such methods are described below.

The disclosure of the present invention of poor NFκB function insidecells of autoimmune mammals implicates decreased resistance of targettissues to such inflammatory cytokine insults.

To evaluate whether a patient is benefitting from treatment, thepatient's symptoms are examined in a quantitative way, such as by theEDSS (Rudick and Goodkin, supra), or decrease in the frequency ofrelapses, or increase in the time to sustained progression, orimprovement in the magnetic resonance imaging (MRI) behavior infrequent, serial MRI studies and compare the patient's statusmeasurement before and after treatment. In a successful treatment, thepatient status will have improved, ie., the EDSS measurement number orfrequency of relapses will have decreased, or the MRI scans will showless pathology.

Preferably, treatment should continue as long as multiple sclerosissymtoms are suspected or observed.

Rheumatoid Arthritis

In rheumatoid arthritis, the main presenting symptoms are pain,stiffness, swelling, and loss of function (Bennett, 1984, “The etiologyof rheumatoid arthritis” in Textbook of Rheumatology, Kelley et al.,eds., W. B. Saunders, Philadelphia, pp. 879-886). The multitude of drugsused in controlling such symptoms seems largely to reflect the fact thatnone is ideal. Although there have been many years of intense researchinto the biochemical, genetic, microbiological, and immunologicalaspects of rheumatoid arthritis, its pathogenesis is not completelyunderstood, and none of the treatments clearly stop progression of jointdestruction (Harris, 1985, “Rheumatoid Arthritis: The clinical spectrum”in Textbook of Rheumatology, Kelley. et al., eds., W. B. Saunders,Philadelphia, pp. 915-990).

TNF-α is present in rheumatoid joint tissues and synovial fluid at theprotein and mRNA level (Buchan et al., 1988, Clin. Exp. Immunol., 73:449-455), indicating local synthesis. Detection of this protein bymethods described herein below (e.g. enzyme immunoassay, EIA, orenzyme-linked immunosorbent assay, ELISA) provides a diagnotic indicatorof arthritis independent of clinical symptoms. In addition,autoantibodies may be quantified as described above.

Analysis of improvement in individual patients following treatment ismade using two separate indices. Firstly, an index of disease activity(IDA) is calculated for each time point according to the method ofMallya and Mace (Mallya et al., 1981, Rheumatol. Rehab., 20: 14-17, thecontents of which are fully incorporated herein by reference) with inputvariable of morning stiffniess, pain score, Richie Index grip strength,ESR and Hgb. The second index calculated was that of Paulus (Paulus etal., 1990, Arthritis Rheum., 33: 477-484, the contents of which arefully incorporated herein by reference) which uses input variables ofmorning stiffness, ESR, joint pain/tenderness, joint swelling, patient'sand Physician's global assessment of disease severity.

Rheumatoid factors may be measured using the rheumatoid arthritisparticle agglutination assay (FAPA, FujiBerio Inc., Tokyo, Japan), inwhich titers of {fraction (1/160)} or greater are consideredsignificant. Rheumatoid factors are measured by ELISA (e.g. using a kitsupplied by Cambridge Life Sciences, Ely, UK).

Hashimoto's Disease (Hypothyroidism)

Symptoms include low levels of circulating thryoid hormone, tiredness,yellow skin discoloration, delayed reflexes, slowed heartrate, witheventual edema leading to coma and death.

Graves's Disease (Hyperthyroidism)

Symptoms include high levels of circulating thyroid hormone,hyperactivity, inability to sleep, thinning hair, irritable bowel andorbital abnormality (protruding eyes).

Vitiligo

This disorder is characterized by melanocyte loss in a characteristicpattern on the body. It is initially diagnosed; as is true of otherautoimmune diseases affecting the skin (see “psoriasis” and “pemphigusvulgaris”, below), tissue biopsy is performed to confirm diagnosis.

Psoriasis

The symptom of psoriasis, also present for visual diagnosis, is scalyskin.

Pemphigus Vulgaris

Symptoms of pemphigus vulgaris include skin peeling and scaling. It,too, is diagnosed visually and by skin biopsy.

In addition, genetic diagnosis of autoimmune disease, which is aneffective means of early diagnosis, is possible for diseases for whichgenetic linkage (pedigree) studies have been performed for large (or,alternatively, small but numerous) families of affected individuals.Early diagnosis may, additionally, be facilitated by the simple assay ofNFκB activity in individuals deemed to be at risk of disease; methods bywhich NFκB are described herein, and include in, vitro DNA/proteinbinding and/or transcriptional activation assays.

In order to ensure the safety of treatments according to the invention,following treatment of arthritis or another autoimmune disease, vitalsigns are recorded at intervals for up to 24 hours followingadministration of the therapeutic agent. Patients are later questionedconcerning possible adverse events before each treatment. Preferably, acomplete physical examination is performed at the time of initialdiagnosis. In addition, patients may be monitored by standard laboratorytests including complete blood count, C3 and C4 components ofcomplement, IgG, IgM and IgA, serum electrolytes, creatinine, urea,alkaline phosphatase, aspartate transaminase and total bilirubin. Urineanalysis may, additionally, be performed.

Prior to testing potential therapeutic compositions and methods on humansubjects, testing is performed in an animal model. It is generallyaccepted by those of skill in the art that results obtained through theuse of animal models are predictive of the efficacy of a given treatmentin a human clinical patient. The following section describes a selectionof animal models which are of use in assessing the efficacy of proposedtreatments of autoimmune disease according to the invention.

Animal Models of Autoimmune Disease

i. Mouse Models

Animal models such as the NOD³ (or, simply, NOD) mouse, which is proneto diabetes, Sjögren's syndrome and hemolytic anemia have alsodemonstrated the importance of the H2 (again, the mouse MHC) genomicregion, in combination with non-H2 genes in autoimmunity. Theinheritance of MHC and MHC-linked genes with minimal recombinations(linkage disequilibrium), together with the fact that most of thesegenes contribute to immune responses, has hampered the identification ofthe genes that underlie autoimmunity. Polymorphisms are abundant in theMHC and are readily detected but the challenge remains to identify thosepolymorphisms that contribute to disease susceptibility and havefunctional consequences, and to define the disease-causing mechanisms.

NOD mice, like humans with type I diabetes, exhibit a phenotype in whichconformationally abnormal forms of class I molecules (which can bedetected with conformationally specific antibodies) are present on thesurface of APCs (Faustrnan et al., 1992, supra). The exit of class Imolecules from the endoplasmic reticulum (ER) of NOD mouse APCs isdelayed, and the presentation of test antigens by these cells ismarkedly impaired in in vitro assays of cytotoxic T cell lysis (Li etal., 1994, supra). Surface class I molecules of NOD mouse APCs can bestabilized by culture at low temperature or by the addition ofallele-specific peptides that presumably occupy the emptypeptide-binding pockets of the class I protein.

Impaired antigen presentation and class I assembly may be essential fordisease expression in diabetes-prone NOD mice and humans. Only NODfemales who progress to hyperglycemia or salivary gland destructionpossess the defect; normoglycemic NOD males, 15% of which developdiabetes, lack the APC defect.

The NOD mouse exhibits a rare MHC haplotype known as H-2⁸ ⁷ , in whichmany polymorphisms are apparent (Hattori et al., 1986, supra; Lund etal., 1990, J. Autoimmun., 3: 289; Prochazka et al., 1987, Science, 237:286; Acha-Orbea and McDevitt, 1987, Proc. Natl. Acad. Sci. U.S.A., 84:2435). For instance, the NOD mouse has a rare Tap1 allele with atranscription defect (Faustman et al., 1991, supra), an uncommon Lmp2allele with a transcription defect, and a unique MHC class II gene atthe I-A locus. The quantitative defect in Tap1 transcription, like theclass I cell surface assembly abnormality, correlates with diseaseexpression in NOD mice, again demonstrating a pattern of gene expressionthat can be influenced by the environment (Huang et al., 1995, Diabetes,44: 1114), gender or noninherited gene phenomena (e.g. somatic generearrangements or changes in gene methlyation pattern). Many of thesegenes have similar promoters and respond in unison to external stimuli.In the case of Tap1 and Lmp2, the genes even share the same promoter inopposing orientations. Therapies based on nonspecific immunostimulation,such as injection with CFA or infection with mouse hepatitis virus,ameliorate diabetes in NOD mice. These treatments also increase the rateof Tap1 transcription, and re-educated or reselected the 7 cellrepertoire so that T cell autoreactivity to class I and syngeneicpeptides is eliminated (Huang et al., 1995, supra). These data suggesttranscription or quantitative issues of gene expression could bedominant in patterns of disease expression.

As in humans, lymphocytic developmental errors are characteristic ofmouse (NOD) and rat (BB; see below) models of Type I diabetes (Shimadaet al., 1996, Diabetes, 45: 71-78; Serreze et al., 1993, Proc. Natl.Acad. Sci. U.S.A., 90: 9625-9629; Li et al., 1994, Proc. Natl. Acad.Sci. U.S.A., 91: 11128-11132). For instance, mature T lymphocytes inperipheral blood, spleen and lymph nodes are markedly absent inautoimmune disease-prone BB animals (Crisa et al., 1992, DiabetesMetaholism Rev., 8: 9-37). As might be expected of an immature lymphoidcell, diabetic lymphocytes in animal and human models demonstratedefective intracellular activation of signal transduction pathways,including responses to TNF, lipopolysaccharides (LPS, which arenon-specific immunostimulants) and signal transduction along themicrotubule-associated protein kinase (MAP kinase) pathway of T cellactivation (Serreze et al., 1993, supra; Rapoport et al., 1993, J. Exp.Med., 177: 1221-1226).

Given the established role of antigen presentation in T cell educationand its impairment in numerous autoimmune diseases in both humans andmice, mutations which contribute to the abnormal antigen presentationand processing in the NOD mouse (made apparent, in part, by alteredclass I assembly and altered presentation of syngeneic peptides) are ofsignificant interest; therefore, the NOD mouse provides a good modelsystem in which genetic and environmental factors influencing autoimmunediseases can be studied. Recently, a mutation in the shared,bidirectional Lmp2/Tap1 promoter has been found to reduce expression ofthese genes in the NOD mouse (Yan et al., 1997, J. Immunol., 159:3068-3080)

ii. The BB Rat

Diabetes-prone BB rats have profound peripheral T lymphocyteimmunodeficiencies and lack a surface maturation molecule or lymphocytesRT6, a member of the src tyrosine kinase family (Elder and Maclaren,1983, J. Immunol., 130: 1723-1731; Rigby et al., 1996, Diabetes, 45:1419-1426; Jackson et al., 1983, Metabolism, 32: 83-86; Woda et al.,1986, J. Immunol., 136: 856-859; Greiner et al., 1986, J. Immunol., 136:148-151).

iii Other Models

Other animal models of autoimmune disease as are known in the art are asfollows:

Experimental autoimmune encephalomyelitis (EAE) in mice and rats servesas a model for multiple sclerosis (M.S.) in humans. It is a CD4⁺ T-cellmediated autoimmune disease that is directed against protein componentsof CNS myelin (Miller and Karpus, supra, 1994). In this model, thedemyelinating disease is induced by administration, typically byinjection, of myelin basic protein (MBP), as described by Paterson, P.Y. (1986, Textbook of Immunopathology, eds. Mischer et al., Grune andStratton, New York, pp. 179-213), McFarlin et al. (1973, Science, 179:487480) and Satoh et al. (1987, J. Immunol., 138: 179-184). B10.PL miceare known to have histopathological and clinical similarities to therelapsing-remitting form of human M.S. (Miller and Karpus, 1994, Immun.Today, 15: 356); these mice develop EAE in response to injection withMBP. EAE is characterized by transient asscending paralysis of theaffected mouse's limbs.

Systemic lupus erythematosis (SLE) is tested in susceptible mice asdisclosed by Knight et al. (1978, J. Exp. Med., 147: 1653). Myastheniagravis (MG) is tested in SJL/J female mice by inducing the disease withsoluble acetyl-cholinesterase receptor (AChR) protein from anotherspecies, as described by Lindstrom et al., (1988, Adv. Immunol., 42:233-284). Arthritis is induced in a susceptible strain of mice byinjection of type II collagen, as described by Stuart et al., (1984,Ann. Rev. Immunol., 42: 233-284). Thyroiditis is induced in mice byadministration of thyroglobulin as described by Maron et al., (1980, J.Exp. Med., 152: 1115-1120). Insulin-dependent diabetes mellitus (IDDM)occurs naturally or can be induced in certain strains of mice.

The contents of the above references relating to animal models ofautoimmune disease are all herein fully incorporated by reference.

NFκB

i. Activation

Rather than treating defects in proteolytic processing at the stage ofthe proteolytic processing, it is possible to target treatment accordingto the invention at the restoration of an important downstream target ofproteasome activation, the transcription factor, NFκB and/or itsdownstream targets.

NFκB is a heterodimeric transcription factor composed of 50 and 65 kDsubunits that belong to the rel family; it is present with inhibitoryfactor IκB in the cytoplasm of most cells (Baeuerle and Henkel, 1994,Ann. Rev. Immunol., 12: 141-179; Verma et al., 1995, Genes Dev., 9;2723-2735). This transcription factor is responsive to cell surfacecytokines, such as tumor necrosis factor α, interleukin-1 andcytoplasmic activation of this factor is required prior to nuclearlocalization. NFκB plays an active role in lymphocytic development andin cell survival (Wang et al., 1996Science, 274: 784-787; Beg andBaltimore, 1996, Science, 274: 782-784; Van Antwerp et al., 1996,Science, 274: 787-789; Arsura et al., 1997, Cell Growth Differ., 8:1049-1059; Liu et al., 1996, Cell, 87: 565-576). In B cells, NFκB isconstitutively expressed (Wu et al., 1996, EMBO J., 15: 4682-4690).Knock-out mice missing Re1A (p65) die before birth, in part, due to adescribed developmental defect of the immune system (macrophages, B andT cells) and massive death of liver cells (Arsura et al., 1997, supra;Beg et al., 1995, Nature, 376: 167-170; Bargou et al., 1997, J. Clin.Invest., 100: 2961-2969). In vitro inhibition of NFκB induces similardevelopmental arrest and death of B cells (Liu et al., 1996, supra).

In the NFκB pathway, it has been observed that phosphorylation andubiquitination work in concert to transmit a message to the nucleus andto activate the cell-cycle genes and proteins in the cytoplasm, thusactivating cell signalling, division, development (e.g.,differentiation) and proliferation; stimulating the the human epithelialHeLa cell line with TNF-α switches on a stress-activated MAP(mitogen-activated protein) cascade that promotes the phosphorylation ofIκBα kinase (Lee et al., 1997, Cell, 88: 213-222). The kinase, in turn,phosphorylates the NFκB inhibitor protein IαBκmarking it forubiquitination. In unstimulated cells, IκB binds to- and inhibits theactivity of NFκB. When ubiquitinated IκB is degraded by the proteasome,NFκB translocates to the nucleus where it activates transcription. As isstated in Hopkin (1997, supra), the combination of two highly specificprocesses, phosphorylation and ubiquitination, has been utilized bycells to control complex signal-transduction pathways precisely. Such amechanism which allows for a rapid return to normal is critical in theactivation and de-activation of molecules such as cytokines, which aresaid to act transiently, as constitutive activation would be cytotoxic.

Cell surface signals on lymphocytes activate NFκB through cascades ofkinases (Verma et al., 1995, supra; Baeuerle and Baltimore, 1996, Cell,87: 13-20). A previous report shows a possible association of NF-κB witha cellular serine kinase, resulting phosphorylation and activation ofNF-κB (Ostrowski et al., 1991, J. Biol. Chem., 266: 12722-12733; Hayashiet al., 1993, J. Biol. Chem., 268: 26790-26795). NFκB also can interactwith cyclin dependent kinases (Cdk), phosphorylation steps regulatingcell cycle progression and conveyance of signals for differentiation andapoptosis. Specifically, Cdk8 or Cdk7 (in combination with cyclins)coordinate the metabolism of differentiated cells with extracellularstimuli and regulate transcriptional activation.

ii Activity in the Nucleus

NFκB and other members of the rel family of protein complexes play acentral role in the transcriptional regulation of a remarkably diverseset of genes involved in the immune and inflammatory responses (Grilliet al., 1993, Int. Cytology, 143: 1-62). For example, NFκB is requiredfor the expression of a number of immune response genes, the Ig-κ lightchain immunoglobulin gene, the IL-2 receptor a chain gene, the T cellreceptor β chain gene, and class I and II major histocompatibilitygenes. In addition, NFκB has been shown to be required for a number ofgenes involved in the inflammatory response, such as the TNF-α gene andthe cell adhesion genes, E-selectin, I-cam, and V-cam. NFκB is alsorequired for the expression of a large number of cytokine genes such asIL-2, IL-6, G-CSF, and IFN-β. Finally, NFκB is essential for theexpression of the human immunodeficiency virus (HIV).

iii. Role in the Cytoplasm

In addition to its role as a transcription factor, NFκB is believedmediate events occurring in the cytoplasm. Subunit p65 bindscyclin-dependent kinases (cdk's), cdc's and other cell cycle activators,which are part of a multiprotein complex; the data presented in Example1, below, demonstrates such binding. These proteins control the cellcycle, differentiation, DNA replication and cell proliferation. It isthought that p50 may have similar binding affinities.

iv Role in Autoimmune Disease

Developmental arrest of lymphocytes has been observed in humans withtype I diabetes; such an arrest often manifests itself as an increase inthe number of CD45RA-naive cells (Faustman et al., 1989, Diabetes 38:1462-1468; Faustman, 1993, Diabete Metab. 19: 446-457; Faustman et al.,1990, J. Autoimmunity, 3: 111-116; Faustman et al., 1991, Diabetes, 40:590-597). Functional assays of antigen presentation and analysis ofsurface antigens on lymphocytes have confirmed the existence of diverseand immature lineages of lymphocytes in type I diabetics (Faustman etal., 1991, Science, 254: 1756-1761; Peakman et al., 1993, Lancet, 342:1296; Peakman et al., 1994, Lancet, 343: 424; Peakman et al., 1994,Diabetes, 43: 712-717).

Regardless of the level at which an autoimmune disease is treatedaccording to the methods of the invention, it is necessary to delivertherapeutic agents in a safe and medically expedient manner. Genetherapy provides one set of methods by which bioactive substances, suchas proteins and nucleic acids, may be delivered in active form to- orsynthesized at their intended sites of action. Gene therapy methods arediscussed in the following section.

Gene Therapy According to the Invention

i. Therapeutic Nucleic Acids

Sequences

A therapeutic gene may be transfected for use in the invention using aviral or non-viral DNA or RNA vector, where non-viral vectors include,but are not limited to, plasmids, linear nucleic acid molecules,artificial chromomosomes and episomal vectors. Expression ofheterologous genes has been observed after injection of plasmid DNA intomuscle (Wolff J. A. et al., 1990, Science. 247: 1465-1468; Carson D.A.et al., U.S. Pat. No. 5,580,859), thyroid (Sykes et al., 1994, HumanGene Therapy, 5: 837-844), melanoma (Vile et al., 1993, Cancer Res., 53:962-967), skin (Hengge et al., 1995, Nature Genet., 10: 161-166), liver(Hickman et al., 1994, Human Gene Therapy, 5: 1477-1483) and afterexposure of airway epithelium (Meyer et al., 1995, Gene Therapy, 2:450-460).

Therapeutic nucleic acid sequences useful according to the methods ofthe invention include those encoding receptors, enzymes, ligands,regulatory factors, and structural proteins. Therapeutic nucleic acidsequences also include sequences encoding nuclear proteins, cytoplasmicproteins, mitochondrial proteins, secreted proteins,plasmalemma-associated proteins, serum proteins, viral antigens,bacterial antigens, protozoal antigens and parasitic antigens.Therapeutic nucleic acid sequences useful according to the inventionalso include sequences encoding proteins, lipoproteins, glycoproteins,phosphoproteins and nucleic acids (e.g., RNAs such as ribozymes orantisense nucleic acids). Proteins or polypeptides which can beexpressed using the methods of the present invention include hormones,growth factors, neurotransmitters, enzymes, clotting factors,apolipoproteins, receptors, drugs, oncogenes, tumor antigens, tumorsuppressors, structural proteins, viral antigens, parasitic antigens andbacterial antigens. The compounds which can be incorporated are onlylimited by the availability of the nucleic acid sequence encoding agiven protein or polypeptide. One skilled in the art will readilyrecognize that as more proteins and polypeptides become identified,their corresponding genes can be cloned into the gene expressionvector(s) of choice, administered to a tissue of a recipient organismsuch as a mammalian tissue (including human tissue), and expressed inthat tissue.

Therapeutic sequences according to the invention may encode productswhich restore proteasome activity; such genes are referred to as being‘upstream’ of NFκB. For example, gene expression constructs encodingproteasome components or associated proteins (e.g. the Lmp2/Tap1 genepair, or Lmp2, Lmp7, Tap1 or Tap2) comprising cDNA sequencesfunctionally linked to the corresponding wild-type transcriptionalregulatory sequences are of use. Genes which restore properubiquitination include those encoding members of the superfamily ofubiquitination-mediating enzymes of the classes E1, E2 and E3; as statedabove, human homologues of the yeast ubiquitination enzymes have beendiscovered, among them the UbcH5 (which functions as an E2) and the MDM2oncoprotein, which acts as a ubiquitin ligase, or E3 (see Honda et al.,1997, supra).

Sequences encoding wild-type NFκB subunits for use in the reconstitutionof missing activity resulting from inactivating mutations in either orboth of p65 and p50; genes encoding these proteins may be administeredaccording to the invention. Genes which might compensate for a loss ofproteasome function to activate NFκB by removing the need forproteasome-mediated cleavage of IκB are also of use, for example, arecombinant NFκB cDNA engineered such that its product can no longer bebound by IκB, as discussed above.

Other genes requiring activation by the proteasome encode apolipoproteinB100 (apoB), transcription factors, e.g. STAT transcription factor orDNA repair factor TFIIH, are also of use.

Genes downstream of NFκB (i.e. those which are under NFκBtranscriptional control) may, themselves be expressed as cDNA constructsin a recipient host; however, this requires a knowledge of alldownstream activation targets of NFκB in cells which are to receivetreatment, as well as designing individual expression constructs foreach such gene and ensuring that they are expressed in the proper ratiosrelative to one another an to other cellular proteins. As stated above,such genes include, but are not limited to, those which encode the Ig-κ,light chain immunoglobulin, the IL2 receptor a chain, the T cellreceptor β chain, class I and II major histocompatibility proteins,TNF-α, E-selectin, I-cam, and V-cam, IL-2, IL-6, G-CSF, and IFN-β.

Nucleic acids of use in the invention include those that encode proteinsfor which a patient might be deficient or that might be clinicallyeffective in higher-than-normal concentration as well as those that aredesigned to eliminate the translation of unwanted proteins. As discussedabove, nucleic acids of use according to the invention for theelimination of deleterious proteins arc antisense RNA and ribozymes, aswell as DNA expression constructs that encode them. Note that antisenseRNA molecules, ribozymes or genes encoding them may be administered to apatient by a method of nucleic acid delivery that is known in the art,such as an in vivo or an ex vivo method, as described below.

Therapeutic genes of use in the invention include those whose productsmay suppress the function of inhibitors or other negative regulators ofproteasome function. One such regulator is the 40 kD-, ATP-dependentprotein mentioned above whose release from the proteasome complexpermits proteolytic cleavage of target proteins to occur. Inactivatingnucleic acid sequences such may encode a ribozyme or antisense RNAspecific for the mRNA which encodes the 40 kD protein or, alternatively,may encode an antibody directed against the 40 kD protein or apolypeptide of like sequence with the site on the proteasome complex towhich the 40 kD protein binds in vivo; such a polypeptide could, ifpresent at several-fold molar excess (e.g. 10-fold or more) over theendogenous proteasome component bound by the 40 kD species, serve as tocompete the inhibitory protein off of it. Note that the 40 kD proteasomeregulator is said to exist as a 250 kD multimer when released (see againWO 95/25533). Japanese patent JP 95121484 discloses a non-functionalmutant of this protein which may be of use to titrate functional 40 kDmolecules away from the proteasome complex.

In addition to the need to suppress the activity of inhibitors ofproteasome function, it may be equally necessary to suppress that ofproteins normally targeted for inactivation by the proteasome. Theseinclude oncogene c-Fos, ornithine decarboxylase, tyrosineaminotransferase, c-myb, HMG-R (a key enzyme of sterol synthesis) andapoB (also activated by proteasomes).

Successful methods for the therapeutic administration of antibodies forthe treatment of autoimmune disease (in this case, rheumatoid arthritis)have been disclosed in U.S. Pat. No. 5,698,195, the contents of whichare herein incorporated by reference.

Ribozymes of the hammerhead class are the smallest known, and lendthemselves both to in vitro synthesis and delivery to cells (summarizedby Sullivan, 1994, J. Invest. Dermatol., 103: 85S-98S; Usman et al.,1996, Curr. Opin. Struct. Biol., 6: 527-533).

Physical Properties and Delivery Vehicles

A nucleic acid of use according to the methods of the invention may beeither double- or single stranded and either naked or associated withprotein, carbohydrate, proteoglycan and/or lipid or other molecules.Such vectors may contain modified and/or unmodified nucleotides orribonucleotides. Examples of some therapeutic nucleic acid sequences areenumerated above. In the event that the gene to be transfected iswithout its native transcriptional regulatory sequences, the vector mustprovide such sequences to the gene, so that it can be expressed onceinside the target cell. Such sequences may direct transcription in atissue-specific manner, thereby limiting expression of the gene to itstarget cell population, even if it is taken up by other surroundingcells. Alternatively, such sequences may be general regulators oftranscription, such as those that regulate housekeeping genes, whichwill allow for expression of the transfected gene in more than one celltype; this assumes that the majority of vector molecules will associatepreferentially with the cells of the tissue into which they wereinjected, and that leakage of the vector into other cell types will notbe significantly deleterious to the recipient mammal. It is alsopossible to design a vector that will express the gene of choice in thetarget cells at a specific time, by using an inducible promoter, whichwill not direct transcription unless a specific stimulus, such as heatshock, is applied.

Delivery of a nucleic acid may be performed using a delivery techniqueselected from the group that includes, but is not limited to, the use ofviral vectors and non-viral vectors, such as episomal vectors,artificial chromosomes, liposomes, cationic peptides, tissue-specificcell transfection and transplantation, administration of genes ingeneral vectors with tissue-specific promoters, etc.

ii. Dosage

Generally, nucleic acid molecules are administered in a mannercompatible with the dosage formulation, and in such amount as will beprophylactically and/or therapeutically effective. When the end product(e.g. an antisense RNA molecule or ribozyme) is administered directly,the dosage to be administered is directly proportional to the the amountneeded per cell and the number of cells to be transfected, with acorrection factor for the efficiency of uptake of the molecules. Incases in which a gene must be expressed from the nucleic acid molecules,the strength of the associated transcriptional regulatory seuqences alsomust be considered in calculating the number of nucleic acid moleculesper target cell that will result in adequate levels of the encodedproduct. Suitable dosage ranges are on the order of, where a geneexpression construct is administered, 0.5- to 1 μg, or 1-10 μg, oroptionally 10-100 μg of nucleic acid in a single dose. It is conceivablethat dosages of up to 1 mg may be advantageously used. Note that thenumber of molar equivalents per cell vary with the size of theconstruct, and that absolute amounts of DNA used should be adjustedaccordingly to ensure adequate gene copy number when large constructsare injected.

iii. Administration

Nucleic acid molecules to be administered according to the inventionalso may be formulated in a physiologically acceptable diluent such aswater, phosphate buffered saline, or saline, and further may include anadjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminumphosphate, aluminum hydroxide, or alum are materials well known in theart. Administration of a nucleic acid molecule as described herein maybe either localized or systemic.

Localized Adminstration

It is contemplated that global administration of a therapeuticcomposition to an animal is not needed in order to achieve a highlylocalized effect. Localized administration of a nucleic acid ispreferably by via injection or by means of a drip device, drug pump ordrug-saturated solid matrix from which the nucleic acid can diffuseimplanted at the target site. When a tissue that is the target oftreatment according to the invention is on a surface of an organism,topical administration of a pharmaceutical composition is possible. Forexample, antibiotics are commonly applied directly to surface wounds asan alternative to oral or intravenous administration, which methodsnecessitate a much higher absolute dosage in order to counter the effectof systemic dilution, resulting both in possible side-effects inotherwise unaffected tissues and in increased cost.

Compositions comprising a therapeutic composition which are suitable fortopical administration can take one of several physical forms, assummarized below:

(i) A liquid, such as a tincture or lotion, which may be applied bypouring, dropping or “painting” (i.e. spreading manually or with a brushor other applicator such as a spatula) or injection.

(ii) An ointment or cream, which may be spread either manually or with abrush or other applicator (e.g. a spatula), or may be extruded through anozzle or other small opening from a container such as a collapsibletube.

(iii) A dry powder, which may be shaken or sifted onto the target tissueor, alternatively, applied as a nebulized spray.

(iv) An liquid-based aerosol, which may be dispensed from a containerselected from the group that comprises pressure-driven spray bottles(such as are activated by squeezing), natural atomizers (or “pump-spray”bottles that work without a compressed propellant) or pressurizedcanisters.

(v) A carbowax or glycerin preparation, such as a suppository, which maybe used for rectal or vaginal administration of a therapeuticcomposition.

In a specialized instance, the tissue to which a therapeutic compositionis the lung. In such a case the route of administration is viainhalation, either of a liquid aerosol or of a nebulized powder of Drugdelivery by inhalation, whether for topical or systemic distribution, iswell known in the art for the treatment of asthma, bronchitis andanaphylaxis. In particular, it has been demonstrated that it is possibleto deliver a protein via aerosol inhalation such that it retains itsnative activity in vivo (see Hubbard et al., 1989, Clin. Invest., 84:1349-1354).

Note that in some cases, the surface in question is internal, forexample, the gastric lining; in such a case, topical application wouldcomprise taking the therapeutic composition via an oral route, whetherin liquid, gel or solid form.

Systemic Administration

Systemic administration of a nucleic acid or other therapeuticcomposition according to the invention may be performed by methods ofwhole-body drug delivery are well known in the art. These include, butare not limited to, intravenous drip or injection, subcutaneous,intramuscular, intraperitoneal, intracranial and spinal injection,ingestion via the oral route, inhalation, trans-epithelial diffusion(such as via a drug-impregnated, adhesive patch) or by the use of animplantable, time-release drug delivery device, which may comprise areservoir of exogenously-produced nucleic acid or other material or may,instead, comprise cells that produce and secrete a therapeutic proteinor other agent (see “Ex vivo therapy”, below). Note that injection maybe performed either by conventional means (i.e. using a hypodermicneedle) or by hypospray (see Clarke and Woodland, 1975, Rheumatol.Rehabil., 14: 47-49).

Systemic administration is advantageous when a pharmaceuticalcomposition must be delivered to a target tissue that iswidely-dispersed, inaccessible to direct contact or, while accessible totopical or other localized application, is resident in an environment(such as the digestive tract) wherein the native activity of the nucleicacid or other agent might be compromised, e.g. by digestive enzymes orextremes of pH.

Nucleic acid constructs of use in the invention can be given in asingle- or multiple dose. A multiple dose schedule is one in which aprimary course of administration can include 1-10 separate doses,followed by other doses given at subsequent time intervals required tomaintain and or reinforce the cellular level of the transfected nucleicacid. Such intervals are dependent on the continued need of therecipient for the therapeutic nucleic acid, the ability of a givennucleic acid to self-replicate in a mammalian cell if it does not becomeintegrated into the recipient's genome and the half-life of anon-renewable nucleic acid (e.g. a molecule that will notself-replicate). Preferably, when the medical needs of the recipientmammal dictate that a nucleic acid or a product thereof will be requiredthroughout its lifetime, or at least over an extended period of time,such as a year or more, a nucleic acid may be encoded by sequences of avector that will self-replicate in the target cells. The efficacy oftransfection and subsequent maintenance of the nucleic acid moleculesmay be assayed either by monitoring the activity of a marker gene, whichmay additionally be comprised by the transfected construct, or by thedirect measurement of either the protein product encoded by the gene ofinterest or the reduction in the levels of a protein the production ofwhich it is designed to inhibit. The assays can be performed usingconventional molecular and biochemical techniques, such as are known toone skilled in the art.

Ex Vivo Therapy

As alluded to earlier, it is possible to administer a therapeuticnucleic acid for use not only in in vivo therapy (i.e., that in which anucleic acid is administered directly to a patient for uptake by- andsubsequent expression in cells in situ) but also in ex vivo therapy(i.e., that in which a nucleic acid is administered to cultured orexplanted cells in vitro, which transfected cells are subsequentlytransplanted into the clinical patient in order to supply a therapeuticproduct). Methods of ex vivo gene therapy are described in detail hereinBy these methods, a plasmid which continues to be maintained in atransformed or transfected cell after such a cell has been administered(e.g. via transplantation) to a multicellular host, such as a mammal,delivers a gene product to that individual. It is contemplated that agene of interest, particularly a therapeutic gene, will be expressed bythe transplanted cell, thereby providing the recipient organismparticularly a human, with a needed RNA (e.g., an antisense RNA orribozyme) or protein.

As discussed above, a cell type may be used according to the inventionwhich is amenable to methods of nucleic acid transfection such as areknown in the art. Such cells may include cells of an organism of thesame species as the recipient organism, or even cells harvested from therecipient organism itself for ex vivo nucleic acid transfection prior tore-introduction Such autologous cell transplants are known in the art.One common example is that of bone marrow transplantation, in which bonemarrow is drawn either from a donor or from a clinical patient (forexample, one who is about to receive a cytotoxic treatment, such as highdoses of ionizing radiation), and then transplanted into the patient viainjection, whereupon the cells re-colonize bones and other organs of thehematopoietic system.

a. Cell Dosage

The number of transfected cells which are administered to a recipientorganism is determined by dividing the absolute amount of therapeutic orother gene product required by the organism by the average amount ofsuch an agent which is produced by a transfected cell. Note thatsteady-state plasmid copy number varies depending on the strength of itsorigin of replication as well as factors determined by the host cellenvironment, the availability of nucleotides and replicative enzymecomplexes, as does the level of expression of the gene of interestencompassed by the plasmid, which level likewise is determined by thestrength of its associated promoter and the availability of nucleotidesand transcription factors in a given host cell background. As a result,the level of expression per cell of a given gene of interest must bedetermined empirically prior to administration of cells to a recipient.

While efficient methods of cell transfection and transplantation areknown in the art, they do not ensure that the transfected cell isimmortal. In addition, the requirements of the recipient organism forthe product encoded by the transgene may change over time. In light ofthese considerations, it is contemplated that cells may be administeredin a single dose or in multiple doses, as needed. A multiple doseschedule is one in which a primary course of administration can include1-10 separate doses, followed by other doses given at subsequent timeintervals required to maintain and or reinforce the cellular level ofthe transfected nucleic acid. Such intervals are dependent on thecontinued need of the recipient for the therapeutic gene product.Preferably, when the medical needs of the recipient mammal dictate thata gene product will be required throughout its lifetime, or at leastover an extended period of time, such as a year or more, the transfectedcells will be replenished on a regular schedule, such as monthly orsemi-monthly, unless such cells are able to colonize the recipientpatient in permanent fashion, such as is true in the case of asuccessful bone-marrow cell transplant.

b. Nucleic Acid Dosage

Provided a nucleic acid vector capable of replication in the transfectedcell is used, the absolute amount of nucleic acid which is transfectedinto cells prior to transplantation is not critical, since in cellsreceiving at least one copy of such a vector, the vector will replicateuntil an equilibrium copy-number is achieved. As a first approximation,an amount of vector equivalent to between 1 and 10 copies thereof percell to be transfected may be used; one of skill in the art may adjustthe ratio of plasmid molecules to cells as is necessary to optimizevector uptake. Of particular used in the invention are vectors ortransfection techniques which result in the stable integration of thegene of interest into the chromosome of the transfected cell, so as toaviod the need to maintain selection for cells bearing the vectorfollowing transplantation into a recipient multicellular organism, suchas a human.

c. Administration of Autologous or Syngeneic Cells

A cell type which is commonly transplanted between individuals of asingle species (or, even, from an individual to a cell culture systemand back to the same individual) is that of hematopoietic stem cells(HSCs), which are found in bone marrow; such cells have the advantagethat they are amenable to nucleic acid transfection while in culture,and are, therefore, well suited for use in the invention. Cultures ofHSCs are transfected with a minimal plasmid comprising an operatorsequence and a gene of interest and the transfected cells administeredto a recipient mammal in need of the product of this gene. Transfectionof hematopoietic stem cells is described in Mannion-Henderson et al.,1995, Exp. Hematol., 23: 1628; Schiffmann et al., 1995, Blood, 86: 1218;Williams, 1990, Bone Marrow Transplant, 5: 141; Boggs, 1990, Int. J.Cell Cloning, 8: 80; Martensson et al., 1987, Eur. J. Immunol., 17:1499; Okabe et al., 1992, Eur. J. Immunol., 22: 37-43; and Banerji etal., 1983, Cell, 33: 729. Such methods may advantageously be usedaccording to the present invention. Administration of transfected cellsproceeds according to methods established for that of non-transfectedcells, as described below.

The transplantation of hematopoietic cells, such as in a bone marrowtransplant, is commonly performed in the art by procedures such as thosedescribed by Thomas et al. (1975, New England J. Med., 292: 832-843) andmodifications thereof Such a procedure is briefly summarized: In thecase of a syngeneic gaft or of a patient suffering from an immunologicaldeficiency, no immunosuppressive pre-treatment regiment is required;however, in cases in which a cells of a non-self donor are to beadministered to a patient with a responsive immune system, animmunosuppressive drug must be administered, e.g. cyclophosphamide (50mg/kg body weight on each of four days, with the last does followed 36hours later by the transplant). Leukemic patients routinely receive a1000-rad midline dose of total-body irradiation in order to ablatecancerous blood cells; this irradiation also has an immune-suppressiveeffect. Following pre-treatment, bone marrow cells (which populationcomprises a small number of pluripotent hematopoietic stem cells, orHSCs), are administered via injection, after which point they colonizethe hematopoietic system of the recipient host Success of the graft ismeasured by monitoring the re-appearance of the numerous adult bloodcell types by the immunological and molecular methods which are wellknown in the art. While as few as 1-10 HSCs are, in theory, able tocolonize and repopulate a lethally-irradiated recipient mammal overtime, it is advantageous to optimize the rate at which repopulationoccurs in a human bone marrow transplant patient; therefore, atransplanted bone marrow sample comprising 10 to 100, or even 100 to1000 HSCs should be administered in order to be therapeuticallyeffective.

It is contemplated that both lymphoid and parenchymal cells,particularly those which are targeted for destruction in autoimmunedisease, are of use in the invention. Such parenchymal include those ofthe islets of Langerhans, the thyroid, the adrenal cortex, muscles,cartilagenous- or other synovial tissue, the kidneys, epithelial tissues(both external and internal, particularly that of the intestinal lumen,lung, heart, liver, kidney, neurons and synovial cells) and the nervoussystem.

In that such cells are meant to either to replace those lost toautoimmune destruction or to provide a pool of autoimmune-resistantcells prior to massive cell death, it is necessary to ensure that suchcells indeed are not susceptible to autoimmune disease. Provided thatearly treatment is undertaken, it is possible to harvest small (or, insome cases, large) numbers of cells of the target tissue directly fromthe patient for transfection and reintroduction; alternatively, cells ofa donor of matching tissue type may be used.

To render the transplanted cells resistant, at least collectively, toimmune rejection by the recipient organism, it is contemplated thattransplanted cells expressing a high level of activated NFκB (a highNFκB “set point”), while still subject to destruction by autoimmune hostlymphocytes, would enjoy the advantage of robust proliferative capacityin order to multiply at a rate surpassing that of cell killing, therebyproviding a long-lived population of therapeutic cells to the recipientorganism. Such cells may be transfected with gene expression constructswhich result in the production of high levels of activated NFκB, or maybe cells obtained from a donor selected for high endogenous NFκBactivity, as may be determined in an in vitro transcription assay orDNA/protein binding assay (as described in Example 2, below) usingprotein extracts drawn from such a donor, which may, itself, be atransgenic mammal.

As an alternative, a procedure has been developed which allows for theshielding of transplanted cells, even those transplanted from a membersof one species to another (see also below, for other such methods). As aprotective measure against viral infection, a mechanism has evolved inthe immune system of vertebrates in which viral proteins being producedwithin the infected cells are broken down into peptides by intracellularproteolytic enzymes. Some of the peptides are enfolded by a particularclass (Class I) of proteins of the major histocompatibility complex(MHC) of genes and are transported to the cell surface, where the viralpeptide/MHC protein complex is displayed as a surface antigen.Circulating cytotoxic T lymphocytes (CTLs) having the appropriatespecificity recognize the displayed MHC Class I antigen as foreign andproceed, through activation and a complex lytic cascade, to kill theinfected cell. The MHC Class I proteins are expressed in essentially allnucleated cells of the body and are a key element in the immune system'sability to distinguish between “self” molecules and “foreign” (non-self)molecules. They can be distinguished from the other class of proteins ofthe major histocompatibility complex of genes, known as MHC Class IIproteins.

Although MHC Class I antigens are a magnificent mechanism for combatinginfection, they also are primarily responsible for the failure oftissues, e.g., cells, organs, or parts of organs, that are transplantedfrom one mammal (donor) to another (host). This rejection of tissue bythe host organism was first observed in mouse skin graft experiments inthe 1950s and was named the transplant reaction. The search for thefactor on donor cells that was evidently recognized and attacked by thehost's immune system led finally to the characterization of the twoclasses of MHC proteins (see, Snell, 1957, Ann. Rev. Micrbiol., 2:439-57).

Recognition of donor MHC Class I antigens as foreign by host CTLs occursnot only where the donor tissue is different from a different species (axenogeneic transplant) but also where the tissues are from a donor ofthe same species as the host (an allogeneic transplant). The specificityof the T cell receptors on CTLs and other T cells that bind to Class Iand Class II antigens is such that a single amino acid difference in thestructure of a MHC antigen can be detected as foreign, leading to animmune response. The MHC proteins are expressed from DNA formed byrearrangement of several gene segments in the MHC loci, leading to ahigh degree of polymorphism in MHC proteins.

A method applicable to inhibiting the rejection of transplanted tissuesmediated by recognition of MHC class I antigens is as follows:Transplanted allogeneic or xenogeneic tissue comprising treating thetransplant tissue with an enzyme capable of cleaving MHC Class Iantigens. Removal of Class I antigens from the donor tissue attenuatesthe extent of the immune response mounted by the host mammal receivingthe transplant. Furthermore, the enzyme treatment is an effectivepreparatory treatment for all tissues intended for transplant, withoutregard to the specific MHC antigens displayed on the donor tissue or thespecificities of the immune system cells of the host.

The method of treating tissues to render them suitable for transplantcomprises incubating the donor tissue with an enzyme capable of cleavingMHC Class I antigens, e.g., in an amount and for a sufficient period toremove sufficient MHC Class I antigens to significantly attenuate thehost's immune response to the donor tissue. Such incubation is performedin a medium which allows both enzymatic cleavage of the surface antigensto proceed, but is still amenable to tissue survival (e.g. aphysiological salt buffer, such as PBS, or a cell-, tissue- or organculture medium, such as are known in the art. Typically the mean celldensity of Class I antigens will be reduced below about 10% of untreatedlevels, preferably below 1%. One such useful enzyme is papain.

The enzyme selected for use in this method must be capable of cleavingMHC Class I antigens, that is, removing a MHC Class I protein/peptidecomplex from the surface of a cell on which it was displayed. Usefulcleavage is that which alters the MHC Class I antigen as displayedsufficiently to avoid interaction with the immune system cells of therecipient mammal; the object of this cleavage step is to removesubstantially all of the extracellular portion of the MHC Class Iantigen from the cell. Any amount of MHC Class I antigen that can beremoved from the donor tissue is helpful in avoiding rejection of thetransplant; however, as a practical matter, removal of as much of theMHC Class I antigens as possible without killing the tissue is desired,e.g. a reduction in MHC Class I density of at least 90% or even as muchas 99% is desirable.

Typically, this is accomplished by bathing the donor tissue in asolution of the enzyme for a period to allow the enzyme to react withthe MHC proteins, e.g., from 20 minutes to 24 hours or more. At highenzyme concentration, incubation of tissues may be for even shorterperiods, so long as the cells of the tissues are not damaged. Ingeneral, a minimum of 75% viability of the tissue cells is required,although 90% viability or more is sought. In order to retard resynthesisof the MHC class I molecules, the enzyme treatment is carried out at theoptimal temperature for enzyme activity, but the treated tissue isthereafter maintained at a low temperature, for example at 4° C., untilready for use.

There are several advantages to the use of enzymes as a treatment foravoiding transplant rejection: (a) the enzymes are comparativelyinexpensive, and many are commercially available in high purity withwell-characterized activity and specificity; (b) enzymes can be usedlocally or in vitro to avoid systemic treatments; (c) enzyme shaving ofthe transplant tissue can be used in combination with (i.e., withoutforeclosing) other complementary therapies; and (d) the use of enzymesis not species-restricted or allelically restricted, and thus the methodis adaptable to veterinary, human and xenogenic tissue treatment withoutradical modification of the procedures or reagents. Since the tissueswill remain viable after treatment, expression of MHC molecules willcontinue, and eventually reappearance of MHC antigens on the donortissue will occur, e.g., after transplantation; consequently, it is thismethod may be used as part of an overall therapy that may includeadditional measures to avoid rejection of the transplanted cells, suchas immunosuppression, plasmaphoresis, antigen blocking, transfection,and the like. Although pre-transplantation treatment of the tissues willbe the most common practice, it is also contemplated that this method ofthe present invention may be employed in situ to effect local immuneresponse inhibition to preserve previously translated tissue. In suchcases, cleavage of the surface antigen produces a local, soluble,competitive receptor for the cells of the host's immune system, whichmay serve to effectively blunt immune attack on the transplanted tissue.

Useful enzynes include proteolytic enzymes, gycosidases, proteinases andcombinations of such enzymes that may sufficiently alter the surfaceantigens to inhibit subsequent transplant rejection. Examples include,but are not limited to, endoproteinase, pepsin, papain, chymotrypsin,trypsin, collagenase, cyanogen bromide, enterokinase (Asp orGlu-specific), iodosobenzoate, lysobacter endoproteinase,N-bromosuccinimide, N-chlorosuccinimide, hydroxylamine,2-nitro5-thiocyanobenzoate and endopeptidase. Papain particularly ofuse, as it is known to cut all MHC Class I molecules of differentalleles and different species in the α3 domain. Papain does not cut theα1 or α2 domain.

Papain cutting characteristics are well described. Papain is the majoringredient of meat tenderizers and is sulfhydryl protease isolated fromthe latex green fruit of papaya It was first isolated in 1955 and itsenzymatic capabilities have been extensively documentated. In its nativestate, the enzyme is inactive, and therefore donor tissue treatments maybe advantageously carried out with a high degree of control, usingnative papain in the presence of activators such as cysteine (0.005 M)and/or EDTA (0.002 M). See generally, Stockell et al., 1957, J. Biol.Chem., 227: 1-26.

Additional such enzymatic reagents include, but are not limited to,oxidoreductases acting on: (1) OH-OH groups: (2) aldehyde or ketogroups; (3) CH-CH groups; (4) CH-NH, groups; (5) reduced NAD or NADP;(6) nitrogenous compounds; (7) diphenols; (8) acting on H₂O₂; (9)hydrogen; (10) acting on single donors with incorporation of oxygen: and(11) acting on paired donors with incorporation of oxygen into onedonor, tranferases: (1) transferring onecarbon groups(methyltranferases, hydroxymethyl-, formyl-and related transferases,carboxyl- and carbamoyltransferases, amidinotransferases); (2)transferring aldehydic or ketonic residues; (3) acting onacyltranferases, aminoacyltransferases); (4) acting onglycosyltranferases (hexosyltranferases, pentosyltranferases); (5)transferring alkyl or related groups; (6) transferring nitrogenousgroups; (7) transferring phosphorus-containing groups(phosphotranferases with an alcohol group as acceptor,phosphotransferases with a carboxyl group as acceptor,phosphotranferases with a nitrogenous group as acceptor,phosphotransferases with a phosphate group as acceptor,phosphotransferases, pyrophosphotransferases, nucleotidyltransferases,transferases for other substituted, phospho-groups); and, (8)transferring sulphur-containing groups (sulphurtransferases,sulphotransferases, CoA-transferases); hydrolases: (1) acting on esterbonds (carboxylic ester hydrolases, thiolester hydrolases, phosphoricmonoester hydrolases, phosphoric diester hydrolases, triphosphoricmonoester hydrolases, sulphuric ester hydrolases); (2) acting onglyeosyl compounds (glycoside hydrolases, hydrolysing N-glycosylcompounds, hydrolysing S-glycosal compounds); (3) acting on ether bonds(thioether hydrolases); (4) acting on peptide bonds (peptide hydrolases)(α-amino-acyl-peptide hydrolases, peptidyl-amino-acid hydrolases,dipetide hydrolases, peptidyl-peptide hydrolases); (5) acting on C—Nbonds other tan peptide bonds (in linear amidines, in cylic amides, inlinear amidines, in cylic amidines, in cyanides); (6) acting onacid-anhydride bonds (in phosphoryl-containing anhydrides); (7) actingon C═C bonds; (8) acting on carbon-halogen bonds; (9) acting on P—N;lyases (1) acting on carbon-carbon bonds (carboxyl-lyases,aldehyde-lyases, keto acid-lyases); (2) acting on carbon-oxygen bonds(hydrolyases and other carbonxygen lyases); (3) acting oncarbon-nitrogen bonds (amonia-lyases and amidine-lyases); (4)carbon-sulphur lyases; (5) carbon-halogen lyases; (6) other lyases;isomerases: (1) racemases and epimerases (acting on amino acids andderivatives; acting on hydroxyacids and derivatives, acting oncarbohydrates and derivatives, acting on other compounds; (2) acting oncis-trans isomerases; (3) acting on intramolecular oxidoreductases(interoconverting aldoses and ketoses, interoconverting keto- andenol-groups, transposing C═C bonds); (4) acting on intramoleculartransferases (transferring acyl groups, transferring phosphoryl groups,transferring other groups); (5) acting on intramolecular lyases; (6)other isomerases; ligases: (1) acting on forming C—O bonds(amino-acid-RNA ligases); (2) acting on forming C-N bonds (acid-ammonialigases (amide synthetases), acid-amino-acid ligases (peptidesynthetases), cyclo-ligases, other C—N ligases, C—N ligases withglutamine as N-donor); (3) forming C—C bonds; and glycosidases, such asα-amylase, β-amylase, glucoamylase, celulase, laminarinase, inulase,dextranase, chitinase, polygalacturonase, lysozymne, neuraminidase,α-glucosidase, β-glucosidase, α-galactosidase, , β-galactosidase,α-mannosidase, β-fructofuranosidase, trehalase, chitobiase,β-acetylglucosaminidase, β-glucuronidase, dextrin-1,6-glucosidase,hyaluronidase, β-D-fucosidase, metalopeptidases, phospholiphase C andnucleosidase.

d. Administration of Xenogeneic and Allogeneic Cells

While transfection and subsequent tranplantation of cells which areobtained from an individual or cell culture system of like species withthe recipient organism may be performed, it is equally true that theinvention may be practised using cells of another organism (such as awell-characterized eukaryotic microorganism, e.g. yeast, in whichappropriate processing of proteins encoded by therapeutic genes islikely and in which useful origins of replication,are known). In such acase, certain concerns must be addressed.

First, when a protein is encoded by the gene of interest, thetransplanted cells must produce the protein in a form that may is of useto the recipient organism. Post-translational processing (including, butnot limited to, cleavage and patterns of glycosylation) must beconsistent with proper function in the recipient. In addition, either aprotein or an RNA molecule of interest must be made available to therecipient after synthesis, such as by secretion, excretion or exocytosisfrom the transplanted cell. To address the former, the protein producedby the transfected cells may be qualitatively compared to the nativeprotein produced by an individual of the same species as the recipientorganism by biochemical methods well known in the art of proteinchemistry. The latter, release of the protein of interest by the cellsto be transplanted, may be assayed by isolating protein from culturemedium which has been decanted from the transfected cells or from whichsuch cells have been separated (i.e. by centrifugation or filtration),and performing Western analysis using an antibody directed at theprotein of interest. Antibodies against many proteins are commerciallyavailable; techniques for the production of antibody molecules are wellknown in the art.

Second, the cells must be shielded from immune rejection by therecipient organism. It is contemplated that such cells may betransfected with constructs expressing cell-surface markers (e.g. MHCantigens) characteristic of the recipient patient so as to provide themwith biochemical camoflage.

In addition, methods for the encapsulation of living cultures of cellsfor growth either in an artificial growth environment, such as in afermentor, or in a recipient organism have been developed, and are alsoof use in the administration of cells transfected according to theinvention. Such an encapsulation system renders the cell invisible toimmune detection and, in addition, allows for the free exchange ofmaterials (e.g. the gene product of interest, oxygen, nutrients andwaste materials) between the transplanted cells and the environment ofthe host organism.

Methods and devices for cell encapsulation are disclosed in numerousU.S. Patents; among these are U.S. Pat. Nos. 4,353,888; 4,409,311;4,673,566; 4,744,933; 4,798,786; 4,803,168; 4,892,538; 5,011,472;5,158,881; 5,182,111; 5,283,187; 5,474,547; 5,498,401 (which isparticularly directed to the encapsulation of bacterial and yeast cellsin chitosan); 5,550,050; 5,573,934; 5,578,314; 5,620,883; 5,626,561;5,653,687; 5,686,115; 5,693,513; and 5,698,413, the contents of whichare fully incorporated by reference herein. Typically required for thesuccessful culture of encapsulated cells is a selectively-permeableouter covering or ‘skin’ which is biocompatible (i.e., tolerated by boththe encapsulated cells and the recipient host), and, optionally, amatrix in- or upon which cells are distributed such that the matrixprovides structural support and a substrate to which anchorage-dependentcells may attach themselves. As relates to encapsulation devicesapplicable to use in the invention, the term “selectively-permeable”refers to materials comprising openings through which small molecules(including molecules of up to about 50,000 M.W.-100,000 M.W.) may pass,but from which larger molecules, such as antibodies (approximately150,000 M.W.), are excluded. Suitable covering materials include, butare not limited to, porous and/or polymeric materials such aspolyaspartate, polyglutamate, polyacrylates (e.g., acrylic copolymers orRL®, Monsanto Corporation), polyvinylidene fluoride, polyvinylidienes,polyvinyl chloride, polyurethanes, polyurethane isocyanates,polystyrenes, polyamides, cellulose-based polymers (e.g. celluloseacetates and cellulose nitrates), polymethyl-acrylate, polyalginate,polysulfones, polyvinyl alcohols, polyethylene oxide, polyacrylonitrilesand derivatives, copolymers and/or mixtures thereof, stretchedpolytetrafluoroethylene (U.S. Pat. Nos. 3,953,566 and 4,187,390, bothincorporated herein by reference), stretched polypropylene, stretchedpolyethylene, porous polyvinylidene fluoride, woven or non-wovencollections of fibers or yarns, such as “Angel Hair” (Anderson, Science,246: 747-749; Thompson et al., 1989, Proc. Natl. Acad. Sci. U.S.A., 86:7928-7932), fibrous matrices (see U.S. Pat. No. 5,387,237, incorporatedherein by reference), either alone or in combination, orsilicon-oxygen-silicon matrices (U.S. Pat. No. 5,693,513). Polylysinehaving a molecular weight of 10,000 to 30,000, preferably 15,000 to25,000 and most preferably 17,000 is also of use in the invention (seeU.S. Pat. No. 4,673,566). Alternatively, the matrix material, comprisingthe transfected cells of the invention, is exposed to conditions thatinduce it to form its own outer covering, as discussed below.

As described in U.S. Pat. No. 5,626,561, the selective permeability ofsuch a covering may be varied by impregnating the void spaces of aporous polymeric material (e.g., stretched polytetrafluoroethylene) witha hydrogel material. Hydrogel material can be impregnated insubstantially all of the void spaces of a porous polymeric material orin only a portion of the void spaces. For example, by impregnating aporous polymeric material with a hydrogel material in a continuous bandwithin the material adjacent to and/or along the interior surface of aporous polymeric material, the selective permeability of the material isvaried sharply from an outer cross-sectional area of the material to aninner cross-sectional area of the material. The amount and compositionof hydrogel material impregnated in a porous polyhrneric materialdepends in large part on the particular porous polymeric material usedto encapsulate cells for transplant. Examples of suitable hydrogelmaterials include, but are not limited to, HYPAN® Structural Hydrogel(Hymedix International, Inc.; Dayton, NJ), non-fibrogenic alginate, astaught by Dorian in PCT/US93/05461, which is incorporated herein byreference, agarose, alginic acid, carrageenan, collagen, gelatin,polyvinyl alcohol, poly(2-hydroxyethyl methacrylate),poly(N-vinyl-2-pyrrolidone) or gellan gum, either alone or incombination.

The matrix typically has a high surface-area:volume ratio, comprisingpores or other spaces in- or on which cells may grow and through whichfluids may pass; in addition, suitable matrix materials are stablefollowing transplantation into a recipient organism. Preferably, thematrix comprises an aggregation of multiple particles, fibers orlaminae. Alternatively, a matrix may comprise an aqueous solution, suchas a physiological buffer or body fluid from the recipient organism (seeU.S. Pat. No. 5,011,472). Suitable matrix materials include liquid,gelled, polymeric, copolymeric or particulate formulations of aminatedglucopolysachharides (e.g., deacetylated chitin, or “chitosan”, which isprepared from the pulverized shells of crabs or other crustaceans and iscommercially available as a dry powder; Cat # C 3646, Sigma, St. Louis,Mo.), alginate (U.S. Pat. No. 4,409,331), poly-β-1→5-N-acetylglucosamine(pGlcNAc) polysaccharide species (either alone of formulated asco-polymer with collagen; see U.S. Pat. No. 5,686,115), reconstitutedextracellular matrix preparations (e.g. Matrigel®; CollaborativeResearch, Inc, Lexington, Mass.; Babensee et al., 1992, J. Biomed. Matr.Res., 26: 1401), proteins, polyacrylamide, agarose and others.

Methods by which cells become encapsulated using such materials are bothnumerous and varied. Encapsulation devices comprising a semi-permeablemembrane material, as described above, may be pre-formed, filled withcells (e.g. by injection or other manual means) and then sealed (U.S.Pat. Nos. 4,892,538; 5,011,472; 5,626,56; and U.S. Pat. No. 5,653,687);such sealing may be effectively permanent (e.g. by the use ofheat-sealing), semi-permanent (e.g. by the use of a biocompatibleadhesive, such as an epoxy, which will not dissolve or degrade in anaqueous environment) or temporary (e.g. by the use of a removable cap orplug, or by shutting of a valve or stopcock). Methods of permanent andsemi-permanent sealing are disclosed in U.S. Pat. No. 5,653,687. As analternative to the use of a pre-formed, semi-permeable cell reservoir,methods by which cells suspended in matrix material and the substancewhich is to form the outer covering of the encapsulation device areco-extruded under conditions which cause the cell/matrix mixture, whichmay be in liquid or semi-liquid (i.e., gelled) form to be encased in acontinuous tube of the semi-permeable polymer, which either forms, orbecomes crosslinked, under the extrusion conditions; such an extrusionprocedure may lead to the formation of capsules which have only one cellreservoir (U.S. Pat. No. 5,283,187) or which are divided into multiple,discrete compartments (U.S. Pat. No. 5,158,881). As an alternative toboth types of procedure, a liquid or semi-liquid (i.e., gelled)cell/matrix mixture droplet is suspended either in an agent whichinduces ‘curing’ or crosslinking of the outer layer of matrix materialto form a semi-permeable barrier (U.S. Pat. Nos. 4,798,786 and5,489,401) or in a solution of polymeric material (or monomers thereof),which will polymerize and/or crosslink upon contact with the cell/matrixdroplet such that a semi-permeable membrane is deposited thereon (U.S.Pat. Nos. 4,353,888; 4,673,566; 4,744,933; 5,620,883; and 5,693,513).

One of such of skill in the art is well able to select the appropriatematrix and semi-permeable membrane materials and to construct acell-encapsulation device as described above.

Implantation of such a device is achieved surgically, via standardtechniques, to a site at or near the anatomical location to which theproduct encoded by the gene on the gene of interest is to be delivered,as is deemed safest and most expedient. Such a device may take aconvenient shape, including, but not limited to, that of a sphere,pellet or other capsule shape, disk, rod or tube; often, the shape ofthe device is determined by its method of synthesis. For example, onewhich is formed by co-extrusion of a cell suspension and a polymericcovering material is typically tubular, while one formed by thedeposition of a covering on droplets comprising cells in matrix materialmight be spherical. As discussed above, the number of cells which mustbe implanted (and, therefore, encapsulated) is dependent upon therequirements of the recipient organism for the product of thetransfected gene. The encapsulation devices described above aretypically small.(most usefully, 10 μm to 1 mm in diameter, so as topermit efficient diffusion of substances back and forth between theouter covering and the cells most deeply embedded in the matrix), and itis contemplated that such devices may carry between and 10 and 10¹⁰cells each. Should the need for larger numbers of cells be anticipated,a plurality (2, 10 or even 100 or more) of such in vivo culturingdevices may be made and implanted in a given recipient organism.

An encapsulated cell device may be intended for permanent installation;alternatively, retrieval of the device may be desirable, whether toterminate delivery of the product of the gene of interest to therecipient organism at the discretion of one of skill in the art, such asa physician (who must determine on a case-by-case basis the length oftime for which a given cell implant is beneficial to the recipientorganism) or to replenish the device with fresh cells after long-termuse (i.e. months to years). To the latter end, an implantation devicemay usefully comprise a retrieval aid, such as a guidewire, and a cap orother port, such as may be opened and re-sealed in order to gain accessto the cell reservoir, both as described in U.S. Pat. No. 4,892,538.

Live cultures of encapsulated cells have been used successfully todeliver gene products to tissues of a recipient animal. U.S. Pat. No.4,673,566 discloses successful maintenance of normal blood sugar levelsin a diabetic rat into which encapsulated rat islet of Langerhans cellswere implanted; two administrations of 3,000 cells each together wereeffective for six months, while a single dose of 1,000 cells waseffective for two months.

Encapsulated GABA-secreting pancreatic cells implanted into subthalamicnucleus of monkeys in whom Parkinsonism has been clinically-induced havebeen observed relieve the symptoms of that syndrome (U.S. Pat. No.5,474,547), demonstrating invisibility of encapsulated cells to theimmune system, as well as efficacy in delivering a product ofencapsulated, transplanted cells to a recipient organism.

More encouraging, as it demonstrates immunological shielding by cellencapsulation systems sufficient for cross-species cell transplants, asis advantageous for their use in practicing the present invention, isthe finding that encapsulated embryonic mouse mesencephalon cells, whentransplanted into recipient rats, alleviate symptoms ofclinically-induced Parkinsonism (U.S. Pat. No. 4,892,538).

Similarly, heterospecific transplantation of encapsulated islet cellshas been demonstrated to treat diabetes successfully (dog islet cells toa mouse recipient, U.S. Pat. No. 5.578,314; porcine islet cells to amouse recipient, Sun et al., 1992, ASAIO J., 38: 124). It is believedthat such an approach is promising for the clinical treatment ofdiabetes mellitus in humans (Calafiore, 1992, ASAIO J., 38: 34).

It is contemplated that these techniques, which have been appliedsuccessfully to untransfected cells, may be utilized advantageously withcells that are transfected with therapeutic nucleic acid molecules ofuse in the invention.

e. Assay of efficacy of transplanted cells in a recipient organism

The efficacy of the transfected cells so administered and theirsubsequent maintenance in the recipient host may be assayed either bymonitoring the activity of a marker gene, which may additionally becomprised by the transfected construct, or by the direct measurement ofeither the product (e.g. a protein) encoded by the gene of interest orthe reduction in the levels of a protein the production of which it (anantisense message or ribozyme) is designed to inhibit. The assays can beperformed using conventional molecular and biochemical techniques, suchas are known to one skilled in the art, or may comprise histologicalsampling (ie., biopsy) and examination of tranplanted cells or organs.

In addition to direct measurements of protein or nucleic acid levels inblood or target tissues encoded by the gene of interest borne by thevector in transfected/transplanted cells, it is possible to monitorchanges in the disease state in patients receiving gene transfer viatransplantation of cells in which the gene of interest is maintained andcompare them to the progression or persistence of disease in patientsreceiving comparable cells transfected with vector constructs lackingthe gene of interest.

Proteins and other Therapeutic Agents

In addition to nucleic acids, proteins and perhaps other bioactivesubstances may be used to stimulate proteosome activity in a recipientmammal. When the amount of a protein or other therapeutic agent to beused is considered, the lowest dose that provides the desired degree ofenhancement of NFκB activity by the target cells should be used; lowerdoses may be advantageous in order to minimize the likelihood ofpossible adverse effects. Note that “NFκB activity” includes not onlythe presence of functional NFκB, but may also include the presence ofthe products of genes regulated by NFκB, regardless of the means bywhich they have arisen in the cell, as well as normal differentiationsproliferation and survival of the cell. It will be apparent to those ofskill in the art that the therapeutically-effective amount of acomposition administered in the invention will depend, inter alia, uponthe efficiency of cellular uptake of a composition, the administrationschedule, the unit dose administered, whether the compositions areadministered in combination with other therapeutic agents, the health ofthe recipient, and the therapeutic activity of the particular protein orother pharmaceutical substance.

As is also true of nucleic acids administered according to theinvention, the precise amount of a protein or other pharmaceutical agentrequired to be administered depends on thejudgmnent of the practitionerand may be peculiar to each subject, within a limited range of values.An appropriate dose of a protein or other substance may be calculated asfollows:

The NOD mouse model may be used to assay the effectiveness of varyingdoses of a protein or other agent in treating an autoimmune diseaseaccording to the invention. For a given therapeutic composition, it isnecessary to establish an approximate range of dosages that are useful,yet relatively safe, in a clinical situation. The NOD mouse model may beemployed to establish a dosage curve prior to use of the invention inhuman subjects. Alternatively, if a pharmaceutical agent usefulaccording to the invention already has been granted regulatory approval,it stands that acceptable upper limits of dosage tolerance for humansand other mammals already will have been established for these drugsprior to testing, as have systemic concentrations useful for otherclinical applications. These known dosages may serve as the basis uponwhich calculations may be made prior to use of the mouse model.

A therpeutic composition may be administered either systemically orlocally. In the general case, a starting dosage to be administeredlocally to cells in the mice equals the optimal systemic concentrationdescribed for a known use of the therapeutic agent. Ideally, such adosage has been established for mice; otherwise, the relevant humandosage is used for the purposes of calculation. As it is not knownwhether the concentration of a particular protein or other agent that isuseful for enhancing NFκB activity is higher or lower than that used forother clinical purposes, a range of values above and below therecommended dosage may be assayed. In a first attempt, values spanningfour orders of magnitude below this dosage are examined; if no effect isseen, or if enhancement of NFκB activity in the target cells is observedto increase at or near the starting dosage, values that exceed thatdosage by up to four orders of magnitude are assayed. If no effect isseen within four orders of magnitude in either direction of the startingdosage, it is likely that the agent is not of use according to theinvention. It is critical to note that when elevated dosages are used,the concentration must be kept below harmful levels, which are alsoknown for all drugs that are approved for clinical use. Such a dosageshould be one (or, preferably, two or more) orders of magnitude belowthe LD₅₀ value that is known for a laboratory mammal, whether or notthat mammal is a mouse, and preferably below concentrations that aredocumented as producing serious, if non-lethal, side effects. If itdetermined that a therapeutic agent is optimally useful at levels thatare harmful if achieved systemically, that agent should be used forlocal administration only, and then only at such doses where diffusionof the drug from the target site reduces its concentration to safelevels.

Assessment of Changes in Proteasome Activity According to the Invention

Methods for assessing proteasome activity following treatment are asdescribed above for use in the detection of deficiencies in proteolyticprocessing.

Assessment of NFκB Activation According to the Invention

The amount of NFκB in cells treated according to the invention may beassessed by methods well known in the art, as described above for thedetection of defects in proteolysis leading to the failure to activateNFκB.

Molecular Methods

i. North Analysis

Molecular methods such as Northern analysis are well known in the art(see Sambrook et al., 1989, Molecular Cloning A Laboratory Manual., 2ndEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

ii. RT-PCR

As an alternative to Northern analysis, reverse transcription/polymerasechain reaction (RT-PCR) may be performed. In the reverse transcription(RT) step of RT-PCR, the RNA is converted to first strand cDNA, which isrelatively stable and is a suitable template for a PCR reaction. In thesecond step, the cDNA template of interest is amplified using PCR. Thisis accomplished by repeated rounds of annealing sequence-specificprimers to either strand of the template and synthesizing new strands ofcomplementary DNA from them using a thermostable DNA polymerase.

1 μg of total RNA and 75 pmol random hexamer primer (e.g., Pd(n)6,supplied by Pharmacia; Piscataway, N.J.) are resuspended in a 10 μvolumewith DEPC-treated water in an RNase-free 0.5 μl tube. This mixture isincubated at 70° C. for minutes and placed on ice for two minutes. Thefollowing reagents are added to the 10 μl reaction; 10 μl (200U) MMLV-RT(Superscript® reverse transcriptase, BRL, Life Technologies,Gaithersburg, M.D.), 4 μl 5× reaction buffer (BRL, Life Technologies,Gaithersburg, Md.), 2 μl 0.1 M. DTT, 1 μl 10 mM dNTP and 1 μl humanplacental RNase inhibitor (10 to 50 units per μl; Boehringer Mannheim,Indianapolis, Ind.). In addition, for each RNA sample a second reactionis prepared except that MMLV-RT was omitted (RT negative control). The19 μl reaction is incubated for 50 minutes at 42° C. in a programmablethermal cycler (such as is manufactured by MJ Research; Watertown.Mass.) and inactivated by heating to 90° C. for 5 minutes. After coolingto 37° C., 1 μl RNase H (3 units per μl;BRL, Life Technologies,Gaithersburg, Md.) is added, the reaction is incubated at 37° C. forminutes, then cooled to 4° C. RNA integrity is confirmed byamplification of a transcript of a constitutively-expressed gene (e.g.,interleukin-2 or G_(αs)); therefore, it is ensured that a negativeresult subsequently observed on a test sample can be ascribed to a lackof that specific mRNA and not to degradation of the pool of mRNA orfailure of the reverse transcription reaction.

The polymerase chain reaction, or PCR, is then performed as previouslydescribed (Mullis and Faloona, 1987, Methods Enzymol., 155: 335-350,herein incorporated by reference). PCR, which uses multiple cycles ofDNA replication catalyzed by a thermostable, DNA-dependent DNApolymerase to amplify the target sequence of interest, is well known inthe art.

Oligonucleotide primers useful according to the invention aresingle-stranded DNA or RNA molecules that are hybridizable to a nucleicacid template to prime enzymatic synthesis of a second nucleic acidstrand. The primer is complementary to a portion of a target moleculepresent in a pool of nucleic acid molecules used in the preparation ofsets of arrays of the invention. It is contemplated that such a moleculeis prepared by synthetic methods, either chemical or enzymatic.Alternatively, such a molecule or a fragment thereof isnaturally-occurring, and is isolated from its natural source orpurchased from a commercial supplier. Oligonucleotide primers are 15 to100 nucleotides in length, ideally from 20 to 40 nucleotides, althougholigonucleotides of different length are of use.

Typically, selective hybridization occurs when two nucleic acidsequences are substantially complementary (at least about 65%complementary over a stretch of at least 14 to 25 nucleotides,preferably at least about 75%, more preferably at least about 90%complementary). See Kanehisa, M., 1984, Nucleic Acids Res. 12: 203,incorporated herein by reference. As a result, it is expected that acertain degree of mismatch at the priming site is tolerated. Suchmismatch may be small, such as a mono-, di- or tri- nucleotide.Alternatively, it may encompass loops, which we define as regions inwhich mismatch encompasses an uninterrupted series of four or morenucleotides.

Overall, five factors influence the efficiency and selectivity ofhybridization of the primer to a second nucleic acid molecule. Thesefactors, which are (i) primer length, (ii) the nucleotide sequenceand/or composition, (iii) hybridization temperature, (iv) bufferchemistry and (v) the potential for steric hindrance in the region towhich the primer is required to hybridize, are important considerationswhen non-random priming sequences are designed.

There is a positive correlation between primer length and both theefficiency and accuracy with which a primer will anneal to a targetsequence; longer sequences have a higher melting temperature (T_(M))than do shorter ones, and are less likely to be repeated within a giventarget sequence, thereby minmizing promiscuous hybridization. Primersequences with a high G-C content or that comprise palindromic sequencestend to self-hybridize, as do their intended target sites, sinceunimolecular, rather than bimolecular, hybridization kinetics aregenererally favored in solution; at the same time, it is important todesign a primer containing sufficient numbers of G-C nuclcotidc pairingsto bind the target sequence tightly, since each such pair is bound bythree hydrogen bonds, rather than the two that are found when A and Tbases pair. Hybridization temperature varies inversely with primerannealing efficiency, as does the concentration of organic solvents,e.g. formamide, that might be included in a priming reaction orhybridization mixture, while increases in salt concentration facilitatebinding. Under stringent annealing conditions, longer hybridizationprobes (of use, for example, in Northern analysis) or synthesis primershybridize more efficiently than do shorter ones, which are sufficientunder more permissive conditions. Stringent hybridization conditionstypically include salt concentrations of less than about 1 M, moreusually less than about 500 mM and preferably less than about 200 mM.Hybridization temperatures range from as low as 0° C. to greater than22° C., greater than about 30° C., and (most often) in excess of about37° C. Longer fragments may require higher hybridization temperatues forspecific hybridization. As several factors affect the stringency ofhybridization, the combination of parameters is more important than theabsolute measure of a single factor.

Primers are designed with these considerations in mind. While estimatesof the relative merits of numerous sequences may be made mentally by oneof skill in the art, computer programs have been designed to assist inthe evaluation of these several parameters and the optimization ofprimer sequences. Examples of such programs are “PrimerSelect” of theDNAStar™ software package (DNAStar, Inc.; Madison, Wis.) and OLIGO 4.0(National Biosciences, Inc.). Once designed, suitable oligonucleotidesare prepared by a suitable method, e.g. the phosphoramidite methoddescribed by Beaucage and Carruthers (1981, Tetrahedon Lett., 22:1859-1862) or the triester method according to Matteucci et al. (1981,J. Am. Chem. Soc., 103: 3185), both incorporated herein by reference, orby other chemical methods using either a commercial automatedoligonucleotide synthesizer or VLSIPS™ technology.

PCR is performed using template DNA (at least 1 fg; more usefully,1-1000 ng) and at least 25 pmol of oligonucleotide primers; it may beadvantageous to use a larger amount of primer when the primer pool isheavily heterogeneous, as each sequence is represented by only a smallfraction of the molecules of the pool, arid amounts become limiting inthe later amplification cycles. A typical reaction mixture includes: 2μl of DNA, 25 pmol of oligonucleotide primer, 2.5 μl of 10×PCR buffer 1(Perkin-Elmer, Foster City, Calif.), 0.4 μl of 1.25 μM dNTP, 0.15 μl (or2.5 units) of Taq DNA polymerase (Perkin Elmer, Foster City, Calif.) anddeionized water to a total volume of 25 μl. Mineral oil is overlaid andthe PCR is performed using a programmable thermal cycler.

The length and temperature of each step of a PCR cycle, as well as thenumber of cycles, is adjusted in accordance to the stringencyrequirements in effect. Annealing temperature and timing are determinedboth by the efficiency with which a primer is expected to anneal to atemplate and the degree of mismatch that is to be tolerated; obviously,when nucleic acid molecules are simultaneously amplified andmutagenized, mismatch is required, at least in the first round ofsynthesis. In attempting to amplify a population of molecules using amixed pool of mutagenic primers, the potential for loss, under stringent(high-temperature) annealing conditions, of products that would onlyresult from low melting temperatures is weighed against the promiscuousannealing of primers to sequences other than the target site. Theability to optimize the stringency of primer annealing conditions iswell within the knowledge of one of moderate skill in the art. Anannealing temperature of between 30° C. and 72° C. is used., Initialdenaturation of the template molecules normally occurs at between 92° C.and 99° C. for 4 minutes, followed by 20-40 cycles consisting ofdenaturation (94-99° C. for seconds to 1 minute), annealing (temperaturedetermined as discussed above; 1-2 minutes), and extension (72° C. for 1minute). Final extension is generally for 4 minutes at 72° C., and maybe followed by an indefinite (0-24 hour) step at 4° C.

Several techniques for detecting PCR products quantitatively withoutelectrophoresis may be advantageously used with the assay of theinvention in order to make it more suitable for easy clinical use. Oneof these techniques, for which there are commercially available kitssuch as Taqman™ (Perkin Elmer, Foster City, Calif.), is performed with atranscript-specific antisense probe. This probe is specific for the PCRproduct (e.g. a nucleic acid fragment derived from an NFκB-induciblegene) and is prepared with a quencher and fluorescent reporter probecomplexed to the 5′ end of the oligonucleotide. Different fluorescentmarkers can be attached to different reporters, allowing for measurementof two products in one reaction. When Taq DNA polymerase is activated,it cleaves off the fluorescent reporters by its 5′-to-3′ nucleolyticactivity. The reporters, now free of the quenchers, fluoresce. The colorchange is proportional to the amount of each specific product and ismeasured by fluorometer, therefore, the amount of each color can bemeasured and the RT-PCR product can be quantified. The PCR reactions canbe performed in 96 well plates so that samples derived from manyindividuals can be processed and measured simultaneously. The Taqman™system has the additional advantage of not requiring gel electrophoresisand allows for quantification when used with a standard curve.

Detection of NFκB-directed transcripts may advantageously be performedin a single tube reaction for reverse transcription of RNA and specificamplification of transcripts of interest. This system utilizes twoenzymes, AMV reverse transciptase to prepare first strand cDNA, and thethermostable Tf1 DNA polymerase for second strand cDNA synthesis andsubsequent DNA amplification, with an optimized single buffer systemthat permits RT-PCR to be performed in one step, simplifying the assayand minimizing the chance for contamination during preparation of aseparate PCR reaction, Commercial kits such as the Access™ RT-PCR system(Promega; Madison, Wis.) conveniently assemble all materials (exceptprimers) necessary to carry out the method in this way. The single-tubeRT-PCR assay according to this technique may be used to assay serum- orother samples.

Alternatively, it is possible to use an enzyme such as rTth polymerase(Perkin Elmer, Foster City, Calif.) that has reverse transcriptaseactivity in the presence of Mn²⁺ and has DNA polymerase function athigher temperatures (Juhasz et al., 1996, BioTechniques, 20: 592-600).Such an enzyme system allows for single tube and single enzyme RT-PCR.PCR product detection has been performed both by polyacrylamide gelelectrophoresis and ethidium bromide stainig and also by performing thePCR reaction in a 96well plate in combination with a fluorescentdetection system such as the one described above. Utilization of such afluorescent detection system in the one-tube system allows for thesimple addition of RNA to a well containing the buffer, enzymes, dNTPs,primers and the detection probe followed by RT-PCR and luminescentreading. The sensitivities of these systems are equal or superior tostandard two-tube methods (Chehadeh et al., 1995, BioTechniques, 18:26-28; Sellner et al., 1992, Nucleic Acids Res., 20: 1487-1490; Juhaszet al., supra), although there is no excess cDNA available foramplification of multiple transcripts.

Alternatively, in situ detection of mRNA transcripts may be performedusing either ‘squashed’ cellular material or to sectioned tissue samplesaffixed to glass surfaces, prepared as described below. Eitherparaffin-, plastic- or frozen (Serrano et al., 1989, Dev. Biol. 132:410-418) sections are used in the latter case. Following preparation ofeither squashed or sectioned tissue, the RNA molecules of the sample arereverse-transcribed in situ. In order to contain the reaction on theslide, tissue sections are placed on a slide thermal cycler (e.g.Tempcycler II; COY Corp., Grass Lake, Mich.) with heating blocksdesigned to accommodate glass microscope slides. Stainless steel orglass (Bellco Glass Inc.; Vineland, N.J.) tissue culture cloning ringsapproximately 0.8 cm (inner diameter)×1.0 cm in height are placed on topof the tissue section. Clear nail polish is used to seal the bottom ofthe ring to the tissue section, forming a vessel for the reversetranscription and subsequent localized in situ amplification (LISA)reaction (Tsongalis et al., 1994, Clinical Chemistry, 40: 381-384).

Reverse transcription is carried out using reverse transcriptase, (e.g.avian myoblastosis virus reverse transcriptase, AMV-RT; LifeTechnologies/Gibco-BRL or Moloney Murine Leukemia Virus reversetranscriptase, M-MLV-RT, New England Biolabs, Beverly, Mass.) under themanufacturer's recommended reaction conditions. For example, the tissuesample is rehydrated in the reverse transcription reaction mix, minusenzyme, which contains 50 mM Tris-HCl (pH 8.3), 8 mM MgCl₂, 10 mMdithiothreitol, 1.0 mM each DATP, dTTP, dCTP and dGTP and 0.4 mMoligo-dT (12- to 18-mers). The tissue sample is, optionally, rehydratedin RNAase-free TE (10 mM Tris-HCl, pH 8.3 and 1 mM EDTA), then drainedthorougly prior to addition of the reaction buffer. To denature the RNAmolecules, which may have formed some double-stranded secondarystructures, and to facilitate primer annealing, the slide is heated to65° C. for 1 minute, after which it is cooled rapidly to 37° C. After 2minutes, 500 units of M-MLV-RT are added the mixture, bringing the totalreaction volume to 100 μl . The reaction is incubated at 37° C. for onehour, with the reaction vessel covered by a microscope cover slip toprevent evaporation.

Following reverse transcription, reagents are pipetted out of thecontainment ring structure, which is rinsed thoroughly with TE buffer inpreparation for amplification of the resulting cDNA molecules.

The amplification reaction is performed in a total volume of 25 μl,which consists of 75 ng of both the forward and reverse primers (forexample the mixed primer pools 1 and 2 of Example 6) and 0.6 U of Taqpolymerase in a reaction solution containing, per liter: 200 nmol ofeach deoxynucleotide triphosphate, 1.5 mmol of MgCl₂, 67 mmol ofTris-HCl (pH 8.8), 10 mmol of 2-mercaptoethanol, 16.6 mmol of ammoniumsulfate, 6.7 μmol of EDTA, and 10 μmol of digoxigenin-11-dUTP. Thereaction mixture is added to the center of the cloning ring, and layeredover with mineral oil to prevent evaporation before slides are placedback onto the slide thermal cycler. DNA is denatured in situ at 94° C.for 2 min prior to amplification. LISA is accomplished by using cycles,each consisting of a 1 -minute primer annealing step (55 ° C.), a 1.5-min extension step (72° C.), and a 1-min denaturation step (94° C.).These amplification cycle profiles differ from those used in tubeamplification to preserve optimal tissue morphology, hence thedistribution of reverse transcripts and the products of theiramplification on the slide.

Amlpified products containing incorporated digoxigenin-11-dUTP aredetected with a modification of the protocol supplied with the Genius 1kit (Boehringer Mannheim Biochemicals; Indianapolis, Ind.), which isbriefly summarized as follows: Following amplification, the oil layerand reaction mix are removed from the tissue sample, which is thenrinsed with xylene. All solutions and reactions are at room temperature.The containment ring is removed with acetone, and the tissue containingthe amplified cDNA is rehydrated by washing three times in approximately0.5 ml of buffer 1 (100 mM Tris-Cl (pH 7.5) and 150 mM NaCl) and thenincubated for 30 minutes in 0.5 ml of buffer 2 (ml blocking reagent perliter of buffer 1) in a humidified chamber. Subsequently, the slidesbearing the tissue samples are rinsed with 0.5 ml of buffer 1 andincubated for 1 hour with a 1:100 dilution of antibody (alkalinephosphatase-conjugated anti-digoxigenin; Boehringer Mannheim) in ahumidified chamber. Excess antibody is rmoved by three washes in buffer3 (100 mM Tris.HCl, 100 mM NaCl, 50 mM MgCl₂, pH 9.5) before theaddition of hte chromogen (nitroblue tetrazolium chloride and5-bromo4chloro-3-indolyl phosphate). The detection reaction is monitoredfor optimal staining (˜10-25 minutes) and stopped by rinsing three timesin buffer 4 (10 mM Tris.HCl, 1 mM EDTA, pH 8.0). The tissues are thendehydrated in a series of graded alcohols and stained with eosin beforecoverslips are applied; negative control slides are also stained at thistime. Samples are then examined by light microscopy and photographed.

Other measures of restored function include testing of cells for normalmitotic activity, cell viability, cell growth, restored differentiation,normal cell cycle progression and increased protection afforded by NFκB.

Immunological Methods

i. Preparation of Antibodies

Either recombinant proteins or those derived from natural sources can beused to generate antibodies using standard techniques, well known tothose in the field. For example, the proteins are administered tochallenge a mammal such as a monkey, goat, rabbit or mouse. Theresulting antibodies can be collected as polyclonal sera, orantibody-producing cells from the challenged animal can be immortalized(e.g. by fusion with an immortalizing fusion partner) to producemonoclonal antibodies.

1. Polyclonal Antibodies.

The antigen protein may be conjugated to a conventional carrier in orderto increases its immunogenicity, and an antiserum to the peptide-carrierconjugate is raised. Coupling of a peptide to a carrier protein andimmunizations may be performed as described (Dymecki et al., 1992, J.Biol. Chem., 267: 4815-4823). The serum is titered against proteinantigen by ELISA (below) or alternatively by dot or spot blotting(Boersma and Van Leeuwen, 1994, J. Neurosci. Methods, 51: 317). At thesame time, the antiserum may be used in tissue sections prepared asdescribed below. The serum is shown to react strongly with theappropriate peptides by ELISA, for example, following the procedures ofGreen et al., 1982, Cell, 28: 477-487.

2. Monoclonal Antibodies.

Techniques for preparing monoclonal antibodies are well known, andmonoclonal antibodies may be prepared using a candidate antigen whoselevel is to be measured or which is to be either inactivated oraffinity-purified, preferably bound to a carrier, as described byArnheiter et al., Nature, 294, 278-280 (1981).

Monoclonal antibodies are typically obtained from hybridoma tissuecultures or from ascites fluid obtained from animals into which thehybridoma tissue was introduced. Nevertheless, monoclonal antibodies maybe described as being “raised to” or “induced by” a protein.

Monoclonal antibody-producing hybridomas (or polyclonal sera) can bescreened for antibody binding to the target protein. By antibody, weinclude constructions using the binding (variable) region of such anantibody, and other antibody modifications. Thus, an antibody useful inthe invention may comprise a whole antibody, an antibody fragment, apolyfunctional antibody aggregate, or in general a substance comprisingone or more specific binding sites from an antibody. The antibodyfragment may be a fragment such as an Fv, Fab or F(ab′)₂ fragment or aderivative thereof, such as a single chain Fv fragment. The antibody orantibody fragment may be non-recombinant, recombinant or humanized. Theantibody may be of an immunoglobulin isotype, e.g., IgG, IgM, and soforth. In addition, an aggregate, polymer, derivative and conjugate ofan immunoglobulin or a fragment thereof can be used where appropriate.

ii. Detection Method.

Particularly preferred immunological tests rely on the use of eithermonoclonal or polyclonal antibodies and include enzyme-linkedimmunoassays (ELISA), immunoblotting and immunoprecipitation (seeVoller, 1978, Diagnostic Horizons, 2: 1-7, Microbiological AssociatesQuarterly Publication, Walkersville, Md.; Voller et al., 1978, J. Clin.Pathol., 31: 507-520; U.S. Reissue Pat. No. 31,006; UK Pat. 2,019,408;Butler, 1981, Methods Enzymol., 73: 482-523; Maggio, E. (ed.), 1980,Enzyme Immunonassay, CRC Press, Boca Raton, Fla.) or radioimmunoassays(RIA) (Weintraub, B., Princple of radioimmunoassays, Seventh TrainingCourse on Radioligand Assay Techniques, The Endocrine Society, March1986, pp. 1-5, 46-49 and 68-78). For analyzing tissues for the presenceor absence of a protein in the present invention, immunohistochemistrytechniques may be used. Tissue samples to be assayed by these methodsare prepared as described below. It will be apparent to one skilled inthe art that the antibody molecule will have to labeled to facilitateeasy detection of a target protein. Techniques for labeling antibodymolecules are well known to those skilled in the art (see Harlour andLane, 1989, Antibodies, Cold Spring Harbor Laboratory, pp. 1-726).

Alternatively, other techniques can be used to detect the targetproteins, including chromatographic methods such as SDS PAGE,isoelectric focusing, Western blotting, HPLC and capillaryelectrophoresis.

Preparation of Histological Samples

Tissue samples intended for use in in situ detection of either RNA orprotein are fixed using conventional reagents; such samples may comprisewhole or squashed cells, or may instead comprise sectioned tissue.Fixatives adequate for such procedures include, but are not limited to,formalin, 4% paraformaldehyde in an isotonic buffer, formaldehyde (eachof which confers a measure of RNAase resistance to the nucleic acidmolecules of the sample) or a multi-component fixative, such as FAAG(85% ethanol, 4%/o formaldehyde, 5% acetic acid, 1% EM gradeglutaraldehyde). Note that for RNA detection, water used in thepreparation of an aqueous component of a solution to which the tissue isexposed until it is embedded is RNAase-free, i.e. treated with 0. 1%diethylprocarbonate (DEPC) at room temperature overnight andsubsequently autoclaved for 1.5 to 2 hours. Tissue is fixed at 4° C.,either on a sample roller or a rocking platform, for 12 to 48 hours inorder to allow fixative to reach the center of the sample.

Prior to embedding, samples are purged of fixative and dehydrated; thisis accomplished through a series of two- to ten-minute washes inincreasingly high concentrations of ethanol, beginning at 60%- andending with two washes in 95%- and another two in 100% ethanol, followedtwo ten-minute washes in xylene. Samples arc embedded in one of avariety of sectioning supports, e.g. paraffin, plastic polymers or amixed paraffin/polymer medium (e.g. Paraplast®Plus Tissue EmbeddingMedium, supplied by Oxford Labware). For example, fixed, dehydratedtissue is transferred from the second xylene wash to paraffin or aparaffin/polymer resin in the liquid-phase at about 58° C., then replacethree to six times over a period of approximately three hours to diluteout residual xylene, followed by overnight incubation at 58° C. under avacuum, in order to optimize infiltration of the embedding medium in tothe tissue. The next day, following several more changes of medium atminute to one hour intervals, also at 58° C., the tissue sample ispositioned in a sectioning mold, the mold is surrounded by ice water andthe medium is allowed to harden. Sections of 6 μm thickness are takenand affixed to ‘subbed’ slides, which are those coated with aproteinaceous substrate material, usually bovine serum albumin (BSA), topromote adhesion. Other methods of fixation and embedding are alsoapplicable for use according to the methods of the invention; examplesof these are found in Humason, G. L., 1979, Animal Tissue Techniques,4th ed. (W. H. Freeman,& Co., San Fransisco), as is frozen sectioning(Serrano et al., 1989, supra).

Assessment of the Efficacy of Disease Treatment According to theInvention

In addition to direct measurements of protein or nucleic acid levels intarget cells resulting from the specific composition administered by themethods of the present invention, it is possible to monitor changes inthe disease state in patients receiving therapy to enhance NFκB activityand compare them to the progression or persistence of disease in controlpatients who are treated with placebos (i.e. apharmaceutically-acceptable carrier without the therapeutic nucleicacid, protein or other agent).

In treating autoimmune diseases according to the invention, it ispossible to deliver one or more of a number of therapeutically-relevantnucleic acids proteins or other substances to cells or a recipientindividual. A sampling of genes and/or proteins that might be of use isprovided above. Following administration of the chosen composition, animproved rate of improvement in diagnostic clinical indicators (e.g.insulin or blood sugar level in the case of diabetes) in those patientsreceiving the therapeutic gene(s), protein(s) or other agent(s) relativeto those who do not is indicative of efficacious disease treatment usingthe methods of the invention.

The progression of autoimmune disorders may be slowed or reversedaccording to the methods of the invention. Treatment of an autoimmunedisorder using the invention may be judged advantageous if the loss oftissue or function thereof in patients so treated is slowed or haltedrelative to untreated control individuals; for example, the p50 and/orthe p65 gene, which encode the p50 and p65 subunits of NFκB, may beadministered in vivo (e.g., by systemic or localized injection) or exvivo into cells which are subsequently transplanted into a clinicalpatient, and the recipient patient monitored for elimination of tissueor functional loss, or a reduction in such loss sufficient to result innoticeable improvement in health.

EXAMPLE 1

In this Example, the role that phosphorylation of NFκBp65 bycyclin-dependent kinase (Cdk) might play in the maturation oflymphocytes in the immune system is assessed, as is the possibility thatthis NFκBp65 activation step links defective lymphocyte development todiabetes in the NOD mouse model.

To demonstrate an association of NFκBp65 with a cell cycle developmentregulator protein involved in Cdk/Cyclin coupling, aglutathione-S-transferase (GST) NFκBp65 fusion protein was utilized inan affinity purification protocol. GST-NFκBp65 fusion proteins, wildtype NFκBp65 or deletion mutants, NFκBp65 Q417 and NFκBp65 C418 wereconstructed and characterized. GST-carboxy-terminal domain (CTD) of RNApolymerase II large subunit fusion proteins were also constructed andutilized as the substrate of kinase assay. Genes encoding thecarboxy-terminal domain (CTD) of RNA polymerase II large subunit oreither wild-type- or mutant NFκB subunit p65 were inserted into thepGEX2T fusion-protein expression vector (Pharmacia; Uppsala, Sweden) bymolecular biology techniques which are well known in the art (seeSambrook et al., 1989, supra). The GST-CTD or GST-NFκBp65 proteins wereexpressed in E. coli strain BL21 (DE3) LysS. Cultures (50 ml) were grownovernight at 37° C.; the next day, the resulting stationary-phasecultures were diluted 1:100 with fresh LB medium containing ampicillin(100 μg/ml) and grown until A₆₀₀=0.6 optical density units (O.D.U.) at30° C. Production of GST-CTD or GST-NFκBp65 fusion proteins encoded bygenes under control of the Ptac promoter was then induced for 3 hourswith isopropyl-thio-P-D-galactoside (IPTG; an inducer which causesderepression of transcription and subsequent GST fusion proteinexpression) at a final concentration of 0.4 mM. The cultures werecollected by centrifugation and the bacterial pellets are resuspended in4 ml PBS (150 mM NaCl, 16 mM Na₂HP0₄,4 mM NaH₂PO₄) with 5 mM DTT. TheGST-CTD and GST-NFκBp65 fusion proteins were purified from the lysate bybinding to glutathione-Sepharose beads (Pharmacia).

Phosphorylation is a common mechanism of regulating proteins involved incell cycle and transcription and CTD of mammals consists of 52 identicalcopies of the heptapeptide sequence Tyr-Ser-Pro-Thr-Pro-Ser(SEQ. IDNO:3). To investigate if phosphorylation of NFκBp65 might be mediatedthrough a cellular protein kinase, we looked for an association ofNFκBp65 with a cellular protein kinase. Nuclear and cytosolic extractswere prepared from a human T-cell lymphoma cell line, Molt-4; note thatthe preparation protocol is identical to that used to prepare proteinextracts from spleen tissue removed from six-week-old male and femaleNOD mice (see below). Cells were harvested, centrifuged for 15 minutesat 3000 rpm, washed in 10 ml of ice-cold PBS and collected bycentrifugation for 15 minutes at 3000 rpm. The pelleted cells wereresuspended in 4 ml of buffer A (10 mM Hepes, pH 7.8; 10 mM KCl; 2 mMMgCl₂; 1 mM DTT; 0.1 mM EDTA; 0.1 mM PMSF) and incubated on ice for 15min. Then 250 μl of 10% Nonidet P-40 solution (Sigma; St. Louis, Mo.)were added and cells were vigorously mixed and incubated for 30 minutesat 4° C. The harvested cells were centrifuged for 15 min at 3000 rpm.The resulting supernatant comprised the cytosolic fiction, and is hereinreferred to as the “cytosol extract”. Pelleted nuclei were resuspendedin 1500 μl of buffer C (50 mM Hepes, pH 7.8; 50 mM KCl; 300 mM NaCl; 0.1mM EDTA; 1 mM DTT; 0.1 mM PMSF; 10% (v/v) glycerol), mixed for 30minutes and centrifuged for 15 minutes at 3000 rpm at 4° C. Thesupernatant obtained at this step contained the nuclear proteins; hence,this supernatant is herein referred to as the “nuclear extract”. Theconcentration of protein was 20 μg/μl.

GST-NFκp65 and GST-CTD were expressed in BL21 pLysS E. Coli cells andpurified by selective absorption to glutathione sepharose beads.GST-NFκBp65 was incubated with Molt-4 cytosolic and nuclear extracts,prepared as described above. Reaction mixtures were washed in PBS. Theprecipitated complexes were then incubated with GST-CTD of RNApolymerase II large subunit in kinase buffer containing γ-[³²P]ATP aspreviously described (Hayashi et al., 1993, J. Biol. Chem., 268:26790-26795; Faustman et al., 1989, Diabetes, 38: 1462-1468).One-fortieth of the input (I) and supernatant (S) fractions and{fraction (1/40)} of the last wash (W) and pellet (P) fractions wereused for in vitro kinase reaction. Protein complexes were collected bybrief centrifugation, washed and then incubated with GST-CTD substratesin kinase buffer containing y-³²P ATP. The products of the in vitrokinase reactions were then analyzed on SDS-PAGE. A protein ofapproximately 90 kD was phosphorylated in the in vitro kinase reaction(FIG. 1A). The 90 kDa phosphorylated protein was dependent upon thepresence of GST-CTD in the reaction mixtures (FIG. 1B). As FIG. 1 shows,both GST-NFκp65/protein complexes associated with cellular proteinkinases which may phosphorylate a CTD. The prime nucleoside analog,5,6-dichloro-1-β-D-ribofuranosylbenzimidazole. (DRB) can inhibit theactivity of cellular kinases (Marciniak and Sharp, 1991, EMBO J., 10:4189-4196). To determine whether the kinase activity ofNFκBp65-associated protein kinase were sensitive to DRB, the ability ofDRB to inhibit the kinase activity of NFκBp65 association protein kinasewas tested by examining the phosphorylation of CTD in the presence ofdifferent concentrations of DRB. In vitro kinase assays were carried outand phosphorylated GST-CTD products were separated by SDS-PAGE andvisualized by autoradiography. Quantitation of the gel shown in FIG. 1Cwith an image analyzer (a BAS 3000 phosphorimager) was plotted on agraph. The concentrations of DRB indicated in the figure were includedin the respective kinase reaction mixtures. From these data, it isapparent that the kinase activities of NFκBp65-associated proteinkinases (cytosolic and nuclear) were sensitive to DRB in adose-dependent manner. The concentrations of DRB required for 50%inhibition of the activity of NFκBp65-associated protein kinase were 10μM (cytosolic) and 1 μM (nuclear) (FIG. 1C).

To confirm that general inhibition of kinase activity was notresponsible for the observed results, the sensitivity ofNFκBp65-associated protein kinase to DRB was also tested with casein,which has multiple phosphorylation sites, as the substrate. There was adifference in the biochemical character of NFκBp65 association proteinkinase between cytosol and nuclear. In that NFκBp65 may associate withdifferent protein kinases in the cytosol and the nucleus (cytosol; bandA and nuclear, band B), the target amino acid residues on the CTDsubstrate molecule were determined by phosphoamino acid analysis (seeBaeuerle and Baltimore, 1996, supra). In brief, ³²P-labeled GST-CTDfusion proteins were eluted from wet gels and precipitated withtrichloroacetic acid, hydrolyzed for 2 hours in 200 μl of 6 M HClboiling constantly at 110° C. and then dried. The samples wereresuspended in formic acid/acetic acid buffer (pH 1.9) and spotted ontoa glass-backed silica gel plate. These samples, along with 2 μl each ofunlabeled phosphoamino acids (phosphoserine, phosphothreonine andphosphotyrosine; Sigma) as internal markers, were analyzed by thin-layerelectrophoresis at pH 1.9. Phosphoamino acids were visualized byautoradiography. The results, shown in FIG.1D revealed that only serineresidues in the CTD were phosphorylated, indicating that serine orserine/threonine kinases may associate with NFκBp65.

To identify the domain on NFκBp65 molecule which is essential for therecognition by cellular serine and/or serine/threonine kinases, in vitrokinase assays were performed using other deletion mutants, GST-NFκBp65Q417 and -C418. In vitro and in vivo studies indicate that the p65subunit of NF-κB is responsible primarily for transcriptional activationby NF-κB, and a potent transactivation domain has been mapped to acarboxyl-terminal region of p65 that is not shared with p50 (Verma etal., 1995, supra; Baeuerle and Baltimore, 1996, supra; Schmitz andBaeuerle, 1991, EMBO J, 10: 3805-3817; Fujita et al., 1992, Genes Dev.,6: 775-787; Kerr et al., 1993, Nature, 365: 412-419; Pazin et al., 1996,Genes Dev., 10: 37-49 33-36). Therefore, it was of interest to determinewhether the transactivation domain of NFκBp65 were sufficient for theactivity of NFκBp65-associated protein kinases, or it other elementswere required. GST-NFκBp65 Q417 represents the deleted transactivationdomain of NF-κBp65, while GST-NFκBp65 C418 is the GST-trans-activationdomain only of NFκBp65. CTD phosphorylation activities were generatedstrongly in in vitro kinase reaction using GST-NFκBp65 C418/nuclearprotein complexes, but kinase activity was only mildly detected inGST-NFκBp65 C418/cytosolic protein complexes (FIG. 1E), suggesting thatthe transactivation domain of NFκBp65 is required for high-affinitykinase binding. The results of signal quantitation of the gel shown inFIG. 1E (again performed using a BAS 3000 phosphorimager) were plottedon a graph. The difference in the binding properties of NFκBp65 toprotein kinases in the cytosol and the nucleus suggests that NFκBp65associates with different protein kinases in these two regions of thecell.

To further characterize the kinase activities of the proteinsrepresented by cytosolic band A and nuclear band B in FIG. 1D, azide ATPUV-crosslinking assays were performed as previously described (Hayashiet al., 1993, supra). In short, ATP affinity-labeling was performed oncomplexes immunoprecipitated by an anti-NFκBp65 polyclonal antibody(Santa Cruz Biotechnology, Inc.; Calif.) or instead on purified GST-NFκBfusion proteins (wild-type and C417). The protein complexes wereincubated with 10 μCi of 8-azide-α[³²P]ATP in kinase buffer at 37° C.for minutes. The samples were placed 5 cm distant from a UV lamp(wavelength=254 nm) and irradiated on ice for 30 minutes. After additionof 10 μl of sampling buffer (2.5% SDS, 0.65 mM DTT, 0.5 M sucrose) forSDS-polyacrylamide gel electrophoresis (SDS-PAGE), the ATP-bindingproteins were separated on a 12.5% SDS-PAGE and visualized byautoradiography.

The results are shown in FIG. 2. ATP-binding proteins of differentmolecular weights were detected in these assays. Single bandsrepresenting 53 kD and 50 kD proteins with ATP binding activities weredetected in the cytosolic and nuclear samples, respectively (FIG. 2).

It is possible that the 50 kD nuclear ATP-binding protein associateswith NFκB and can phosphorylate CTD, because the observed ATP bindingproteins appeared as single bands in the in vitro ATP-binding assay. Thecytosolic 53 kD protein recognized both wild-type and deletion mutantC418 NFκBp65 proteins and phosphorylated CTD. To verify these findings,similar ATP binding assays were performed with protein complexesimmunoprecipitated from cytoslic and nuclear extracts using ananti-NFκBp65 polyclonal antibody. The proteins UV-crosslinked with8-azide-α-[³²P]ATP were separated by SDS-PAGE and visualized. The 53 kD(cytosolic) and 50 kD (nuclear) protein bands were again detected.Co-immunoprecipitation of these proteins with the NFκBp65 from cytosolicand nuclear extracts metabolically labeled with [³²S]-methionone and[³⁵]-cysteine was also attempted; the results suggest that serine orserine/threonine kinases of 53 kD (cytosolic) and 50 kD (nuclear) canassociate with NF-κBp65 (data not shown).

HIV-1 Tat protein is a trans-activator that selectively activatestranscription in vivo and in vitro experiments. Recent in vitro studiesindicate that Tat activates the CTD phosphorylation activity (Parada andRoeder, 1996, Nature, 384: 375-378). The ability of Tat to activate CTDphosphorylation activities of the protein kinases associated withNFκBp65 was tested by in vitro kinase assays in which the concentrationof Tat in reaction mixture was varied. As shown in FIG. 3, CTDphosphorylation was activated strongly by wild-type Tat in adose-dependent manner; however, no such activation was observed when theTat mutant proteins K41A or Cys22 were assayed. Signal quantitation ofthe gel shown in FIG. 3A was performed as described above, and theresults were plotted on a graph (FIG. 3B). When wild-type Tat wasincluded in the kinase reaction mixture, CTD phosphorylation by thenuclear kinase associated with NFκBp65 was induced 15- and 25-fold morestrongly than in the absence of Tat. On the other hand, CTDphosphorylation activity of the cytosol kinase associated with NFκBp65was activated 10-fold by wild-type Tat. A phosphorylated protein with amolecular mass of approximately 40 kD (again, as judged byelectrophoretic mobility in SDS-PAGE analysis) was detected in thenuclear kinase reaction (FIG. 3A, lane 11). It is likely that Tatprotein was phosphorylated by NFκBp65-associated nuclear kinase, howeverthe phosphorylated form of Tat protein was not detected in the cytosolreaction mixture. This was confirmed by the finding that differentkinases associate with NFκBp65 in the nucleus and the cytosol.

One may conclude that it is likely that serine or serine/threoninekinases with apparent molecular masses of 53 kD (cytosol) and 50 kD(nucleus) associate with NFκBp65. Furthermore, the activities of thesekinases is inhibited by DRB in a dose-dependent manner. These proteinkinases are similar in size to an NFκB kinase which may be cytosolicCdk8 (Tassan et al., 1995, Proc. Natl. Acad. Sci. U.S.A., 92: 8871-8875;Leclerc et al., 1996, Mol. Biol. Cell, 7: 505-513; Rickert et al., 1996,Oncogene, 12: 2631-2640). To determine whether the 53 kD protein kinaseis Cdk8, Western analysis was carried out using an anti-Cdk8 polyclonalantibody and appropriate control antibodies. As shown in FIG. 4A,anti-Cdk8 antibody recognized the 53 kD cytosolic protein kinaseassociated with wild-type and C418 mutant NFκBp65; in addition, Cdk8 wasco-immunoprecipitated with anti-NFκBp65 (FIG. 4C). Cdk2 was found toassociate only weakly with GST-NFκBp65; however, it complexed stronglywith GST-NFκBp65 C418 mutant protein (FIG. 4B). Cdk2 wasco-immunoprecipitated weakly from cytosolic extracts when ananti-NFκBp65 polyclonal antibody was used (FIG. 4C). As expected,TAF_(II)250 and SP1 did not associate with either wild-type or C418mutant NF-κBp65 (FIGS. 4A and 4B). No antibody binding to proteins ofnuclear extracts was observed if wild-type GST-NFκBp65 protein wasadded. Cdk8 and Cdk2 did not co-immunoprecipitate from nuclear extractswith NFκBp65 (FIG. 4C). From these immunoblotting results, it isapparent that cytosolic Cdk8 can associate with NFκBp65; however, theassociation of nuclear Cdk2 NFκBp65 was not clearly indicated.

Type I diabetic models of autoimmunity include a murine model, the NODmouse. As described above, NOD mice exhibit immature forms of T cells, Bcells and macrophages in the immune system as well as signaltransduction errors. To determine whether NFκBp65 dysfunction plays arole in autoimmune pathogenesis, cytosolic and nuclear extracts fromnormal and NOD mice were compared in in vitro kinase assays. The mice at5-6 weeks of age were normoglycemic (hyperglycemic onset due to completeβ cell destruction typically occurs beyond 20 weeks). Cytosolic extractswere prepared from spleens removed from normal mice and NOD mice asdescribed above and elsewhere (Wu et al., 1996, supra). The GST-NFκBp65fusion proteins were mixed with cytosol and nuclear extracts purifiedfrom normal mice and NOD mice. The protein complexes were isolated byaffinity binding to GST-Sepharose beads; after washing, the complexeswere incubated with GST-CTD substrate in a kinase buffer that includedγ-[³²P]ATP. In vitro kinase assay was performed using the CTD substrateand the reaction products were analyzed by 12% SDS-PAGE. Kinase activitywas observed in normal mice, both male and female, but was notdetectable in NOD mice (FIG. 5A). To verify this result, a moresensitive in vitro kinase assay was performed. Rather than using crudeextracts, enriched protein samples were generated using an anti-NFκBp65polyclonal antibody to immunoprecipitate NFκBp65 comlexes from cytosolicand nuclear extracts prepared from normal and NOD mice.NFκBp657-associated kinase activities were strongly evident in normalmice; still no kinase activity was detected in NOD mice (FIG. 5B).

To further characterize the NOD defect in NFκBp65 phosphorylation, thespecific interactions of Cdks with NFκBp65 were evaluated by Westernanalysis. Cdk8 was detected by a polyclonal antibody directed againstit, and was appropriately associated with GST-NFκBp65 in cytosolicextracts derived from normal mice, while no association of NFκBp65 withthe Cdk8 protein was observed in NOD mice (FIG. 5C). In controlextracts, Cdk2 proteins were detected weakly in GST-NFκBp65/proteincomplexes (FIG. 5C); in NOD mice, Cdk2 proteins were not associated withNFκBp65. As expected, TAF_(II)250 and SP1 were not associated withNFκBp65 in control or NOD mice (FIG. 5C, D). No antibody tested was ableto bind proteins found in nuclear extracts of either normal or NOD mice,if wild-type GST-NFκBp65 were added; in this case, neither Cdk8 nor Cdk2was found to co-immunoprecipitate with NFκp65 in nuclear extractsprepared from either mouse strain (FIG. 5C). The basal expression levelsof Cdk8, Cdk2, NFκBp65, SP1 and TAF_(II)250 did not differ betweennormal and NOD mice (data not shown). From these immunoblotting results,it is evident that in cytosolic Cdk8 can associate with NFκBp65 innormal, but not NOD, mice.

EXAMPLE2

In the previous Example, the cytoplasmic activities of NFκB wereexamined. In the present Example, the nuclear activity of NFκB isexplored in both normal and autoimmune mice.

Specific binding of NFκB to its recognition sequence on a nucleic acidmolecule was assayed by electrophoretic mobility shift analysis (EMSA).In this procedure, protein samples are incubated with labeled nucleicacid molecules under conditions which permit nucleic acid/proteinbinding for a time sufficient to allow such binding to occur and thenelectrophoresed on non-denaturing polyacrylamide gels, which aresubsequently subjected to a signal detection procedure, such asautoradiography. AκB binding site consists of a 5′ and a 3′ half site,of which may variants exists; different members of the κB protein family(e.g. NFκB, IκB) have different degrees of affinity for differenthalf-sites or combinations thereof, as reviewed by Siebenlist et al.(1994, supra). In this set of experiments, the affinity of NFκB for twobinding site variants (denoted κB₁ and κB₂) is examined. Lung extractswere used because lung tissue has a high concentration oflung-antigen-presenting cells, and thus would be expected to have highlevels of active NFκB. BALB/c mice display high levels of NFκB activityin the lymphoid cells of the lung. Nuclear extracts and cytosolicextracts were prepared from human T-cell lymphoma Molt4 cells, as wellas lung and spleen tissue removed from six-week-old BALB/c (normal) andNOD autoimmune mice, both male and female. Lung, spleen and culturedcells were harvested, centrifuged for 15 minutes at 3000 rpm, washed in10 ml of ice-cold PBS and collected by centrifugation for 15 minutes at300 rpm. The pelleted cells were resuspended in 4 ml of buffer A (10 mMHepes, pH 7.8; 10 mM KCl; 2 mM; MgCl₂; 1 mM DTT; 0.1 mM EDTA; 0.1 mMPMSF) and incubated on ice for 15 minutes. Then 250 μl of 10% NonidetP-40 solution (Sigma) were added and cells were vigorously mixed andincubated for minutes at 4° C. The harvested cells were centrifuged for15 minutes at 3000 rpm. After centrifugation, the supernatant comprisedcytosolic proteins, and was termed the cytosolic extract. Pelletednuclei were resuspended in 1500 μl of buffer C (50 mM Hepes, pH 7.8; 50mM KCl; 300 mM NaCl; 0.1 mM EDTA; 1 mM DTT.; 0.1 mM PMSF; 10% (v/v)glycerol), mixed for 30 minutes and centrifuged for 15 min at 300 rpm at4° C. This supernatant contained the nuclear proteins at a concentrationof 20 μg/μl), and was termed the nuclear extract. The nuclear lungextracts so prepared were incubated with a ³²P end-labeledoligonucleotide containing the NFκB binding sequence(5′-GATCTAGGGACTTTCCGCTGGGGACTTTCCAG-3′ [SEQ ID NO: 1]) under conditionswhich permit specific DNA/protein binding (e.g., as below). FIG. 6Apresents the results of this experiment (BALB/C male, lanes 2-3 andfemale, lanes 4-5; NOD male lanes 6-7 and female lanes 8-9; Molt-4, laneI0). The labeled DNA probe was included in the reaction mixturescontaining nuclear extracts (1.5 μl lane 2, 4, 6, 8; 3.0 μl lane 3, 5,7, 9, 10) and, as a negative control, in a reaction mixture that wasfree of nuclear extract (lane 1).

As shown in FIG. 6B, the sequence-specificity of NFκB DNA binding wasdetermined in a competitive binding experiment. Nuclear extracts wereincubated with a labeled probe and a molar excess of unlabeled DNA(“cold competitor” or C.C.). Lung tissue nuclear extracts (BALB/C, leftpanel; NOD right panel) were premixed with cold competitor DNA andincubated for 15 minutes on ice before the labeled nucleic acid probewas added; the two competitor sequences were wild-type sequence κB₁:(5′-GATCTAGGGACTTTCCGCTGGGGACTTTCCAG-3′ [SEQ ID NO: 1]) was run in lanes3, 6, 13 and 16, while wild-type sequence κB₂:(5′-GATCTCAGGGGAATCTCCCTCTCCTTTTATGGGCGTAGCG-3′ [SEQ ID NO: 2]) was runin lanes 4, 7, 14 and 17. Nuclear extracts not pre-incubated with coldcompetitor were run in lanes 2, 5, 8, 9, 10, 12, 15, 18, 19 and 20. Thebinding reactions were performed at 37° C. for minutes in a total volumeof 10 μl of buffer containing: 10 mM Hepes (pH 7.9), 50 mM KCl, 5 mMTris-HCl (pH 7.0), 1 mM DTT, 15 mM EDTA, 10% (v/v) glycerol, 1.0 μg ofpoly(dI.dC) and 4 ng of the labeled probe. The DNA-protein complexeswere resoled on nondenaturing 8% polyacrylamide gels. Electrophoresiswas performed with 0.5×TBE buffer (4.5 MM Tris-HCl, 4.5 mM boric acid,0.1 mM EDTA) at 4° C. Again, a negative control containing no nuclearextract was run in lane 1.

The results of NFκB DNA binding experiments using lung cytosolicextracts are presented in FIG. 6C. As shown, cytosolic NFκB/I κBcomplexes were identified by EMSA after treatment of cytosolic extractswith 0.8% DOC (deoxycholate) and 1.2% NP-40. Cytosol extracts wereprepared as described above from BALB/C (lanes 2-5) and NOD (lanes 6-9)mice and Molt-4 cells (lanes 10-11). These extracts either were (“+”,lanes 3, 5, 7, 9 and 11) or were not (“−”, lanes 2, 4, 6, 8 and 10)pretreated with the detergents. As above, a negative control reaction towhich no extract was added was run in lane 1.

The DNA-binding activities of transcription factors other than NFκB werethen assayed; the results of this experiment are shown in FIG. 6D. Thebinding activities were examined by EMSA using as probe an ³²Pend-labeled oligonucleotide containing the SP1 recognition/bindingsequence (left panel) or the AP1 recognition/binding sequence (rightpanel). The respective DNA probes were incubated with nuclear extractsprepared from lung tissue of BALB/C (male, lane 2; female, lane 3) andNOD (male, lane 4; female, lane 5) mice and Molt4 cells (lane 6); anegative control containing no -nuclear extract was run in lane 1. Inall panels, protein-DNA complexes were indicated by arrowheads. (M=male;F=female)

As shown in FIG. 6A, nuclear extracts from the NOD mouse do not exhibitNFκB binding activity to a ³²P-end-labeled probe; these data indicatethat NFκB activity is virtually absent in NOD mice. The data in FIG. 6Bconfirm the specificity of NFκB binding to the labeled probe shown inFIG. 6A, since the cold competitive DNA prevented specific binding ofprotein from the lung extract of BALB/c control mice to the labeledoligonucleotide. The failure to detect active NFκB in either nuclear orcytoplasmic extracts in the NOD mouse indicate that the phenotype isbased upon a deficiency in the activity upstream of the transport ofNFκB to the nucleus. The integrity of the protein extracts derived fromthe NOD mice were confirmed in the experiment shown in FIG. 6D, in whichthe DNA binding capabilities of other transcription factors were assayedand demonstrated to be present; since the DNA-binding activity of twoother lymphocyte-expressed transcription factors, SP1 and AP1, werefound in the NOD mouse extracts, the observed deficiency appears to bespecific to NFκB activation.

Another way of examining transcription factors and their activity is tobind antibodies to the factors and run the complexes on a gel. If thefactor is present, the antibody will bind and thus delay migration downthe gel; such a procedure is known as a “super-shift” assay. In theexperiment shown in FIG. 7A, a labeled DNA probe containing a κB bindingsequence was incubated with nuclear extracts prepared from lung tissueof BALB/c (lanes 1-4) and NOD (lanes 5-8) mice and Molt-4 cells (lanes9-10). Nuclear extracts were pre-incubated either with- (“+”, evennumbered lanes) or without (“−”, odd numbered lanes) an anti-p50polyclonal antibody, and then the labeled DNA probe was added to thereaction mixture.

The results of a similar experiment, this time using an anti-p65polyclonal antibody, are presented in FIG. 7B. Again, nuclear extractsfrom lung tissue of BALB/c (lanes 1-4) and NOD mice (lanes 5-8) andMolt4 cells (lanes 9-10) were pre-incubated either with- (“+”, evennumber lanes) or without (“−”, odd number lane) antibody, and thelabeled DNA probe was then added to the reaction mixture.

This experiment was repeated using an antibody directed against theCCAAT-Box Enhancer Binding Protein (C/EBP); the identities and treatmentof reaction mixtures, as well as their positions on the gel shown inFIG. 7C are otherwise the same as those presented in FIGS. 7A and 7B. Inall cases, DNA/NFκB complexes (NF-κB) and super-shifted DNA-proteincomplexes (S-NF-κB) are indicated by arrows. Nuclear extracts wereprepared from both males (M) and females (F).

Taken as a whole, the data presented in FIG. 7 demonstrate that bothmale and female BALB/C mice possess the activated p50 subunit of NFκB incell nuclei. In contrast, while the activated p50 subunit is virtuallyabsent from the NOD mouse, another active subunit of NFκB is present innuclei obtained from tissue obtained from this autoimmune strain. Whenthis assay was repeated with a p65 antibody to nuclear extracts of NODand BALB/c mice, some p65 was detected in the NOD mouse lung nuclearextract. Since this antibody recognizes both the active and inactiveforms of p65 we cannot tell from this assay if the reduced amounts ofp65 in the nucleus of the NOD were active or inactive. The supershiftadditionally shows that female NOD mice displays a more extremereduction in p65 subunits than does the male, while the BALB/c mouseextracts produce a greater amount of antibody-mediated shift than isobserved with either gender of autoimmune mutant.

FIG. 8A shows the results of experiments to examine the activation ofNFκB nucleic acid-binding by TNF-α treatment. TNF-α is an extracellularsignalling molecule which is thought to upregulate NFκB activation invivo. In order to assess the influence of TNF-α on NFκB in an in vitrosystem, a DNA/protein binding assay was undertaken. Nuclear extractswere prepared from BALB/C and NOD mice by methods described above. Thebinding activities were examined by EMSA with ³²P end-labeledoligonucleotide containing an NFκB recognition sequence. Spleen cellswere stimulated with TNF-α treatment (10 ng/ml;+) or without TNF-αtreatment (−) and nuclear extracts from the treated cells were prepared4 hours later. Nuclear extract prepared from spleen cells of BALB/C(lanes 2-5; FIG. 8A) and NOD (lanes 6-9; FIG. 8A) mice and Molt-4 cells(lanes 10 and 11; FIG. 8A) were incubated with DNA probe as follows:

Double-stranded κB wt, κB mut or IL-R2α κB oligonucleotides wereend-labeled using [α-³²P]dCTP and Klenow polymerase. Binding reactionsof the DNA probe with nuclear extracts were performed at 37° C. forminutes in a total volume of 10 μl of buffer containing 10 mM Hepes (H7.9), 50 mM KCl, 5 mM Tris-HCl (pH 7.0), 1 mM DTT, 15 mM EDTA, 10% (v/v)glycerol, 1.0 μg of poly (dI.dC), and 4 ng of the labeled probe. TheDNA-protein complexes were resolved on nondenaturing 8% polyacrylamidegels. Electrophoresis was performed with 0.5×TBE buffer (4.5 mMTris-HCl, 4.5 mM boric acid, 0.1 mM EDTA) at 4° C. A negative bindingcontrol, to which no nuclear extract was added, was run in lane 1.

In contrast to the results obtained with MOLT-4 and BALB/c cells, TNF-αtreatment only slightly induced NF-κB activation in spleen cells fromNOD mice (male and female) at 10 ng/ml (FIG. 8A). To determine whetherthis effect was concentration-dependent, spleen cells from male andfemale NOD and BALB/c mice were incubated for 4 hours in the absence (−)or presence (+) of TNF-α at 10 ng/ml (FIG. 8B, lanes 3,5,7, and 10) or25 ng/ml (FIG. 8B, lanes 8 and 11). Nuclear extracts were then preparedand assayed for NF-κB DNA-binding activity by EMSA with the KB1oligonucleotide. Lane 1 corresponds to a negative control in which nonuclear extract was added to the reaction mixture. Data represents a gelexposed for 4 days. NF-κB DNA-binding activity detected in TNF-α-treatedBALB/c control spleen cells appeared specific. Various oligonucleotidesin cold competition experiments prevented DNA binding activity of NF-κBto radioactive oligonucleotide probe (data not shown). TNF-α treatmenthad little effect on NF-κB activation in spleen cells from NOD mice atincreasing TNF-α concentrations of 10 or 25 ng/ml (FIG. 8B), with theinduced activity far less than control BALB/c cells, even at 25 ng/ml(FIGS. 8A and 8B).

NFκB DNA binding activity was examined in cytosolic extracts (FIG. 8C).Cytosolic NFκB/I κB complexes were identified by EMSA after treatment ofcytosolic extracts by 0.8% DOC and 1.2% NP-40. Cytosolic extracts thatwere not treated with TNF-α were prepared from spleen cells from BALB/C(lanes 2-5) and NOD (lanes 6-9) mice and Molt-4 cells (lanes 10-11).Cytosolic extracts were either pre-treated (“+”, lanes 3, 5, 7, 9 andI1) or not pre-treated (“−”, lanes 2, 4, 6, 8 and 10) with thedetergents. A reaction to which no extract was added was run as anegative control (lane 1). NF-κB DNA-binding activity in cytosolicextracts of NOD spleen cells was not clearly detected compared to BALB/cmouse spleen cells (FIG. 8C).

In addition to NFκB, other transcription factors were examined for DNAbinding capability in the NOD mouse model in comparison with thatobserved in normal mice. The binding activities were examined by EMSAwith a ³²P end-labeled oligonucleotide containing an SP1 recognitionsite (FIG. 8D, left) or an AP1 recognition site (FIG. 8D, right).Transcription factors SP1 and AP1 had DNA binding activities that didnot differ between BALB/c and NOD extracts (FIG. 8D). Appropriate DNAprobes were incubated with nuclear extract prepared from lung of BALB/c(male, lanes 2, 3 and 11; female, lanes 4, 5 and 12) and NOD mice (male,lanes 6, 7 and 13; female, lanes 8, 9 and 14) and Molt-4 cells (Cane15); again, a negative control reaction, to which no DNA probe wasadded, was run in lanes 1 and 10. In each of FIGS. 8A through 8D,protein-DNA complexes are indicated by arrowheads. Nuclear extracts wereprepared from spleen cells derived from BALB/C or NOD mice. M=male;F=female.

As FIG. 8A clearly shows in spleen cell extracts, TNF-α is only able toactivate NFκB in the BALB/c mouse and in the Molt-4 lymphoid cell line;NOD mice do not show increased p65 activity, suggesting a disruption ofnormal intracellular signalling pathways of p65-mediated protection fromTNF-κ stimulation.

In order to confirm the identities of nuclear proteins binding to κBsites following TNF-α stimulation, a super-shift assay was performed(FIG. 9). A labeled DNA probe containing a κB binding sequence wasincubated with nuclear extracts prepared from spleen cells after TNF-αtreatment. Spleen cells were pre-stimulated by TNF-α treatment for 4hours. Nuclear extracts were preincubated with an anti-p50 polyclonalantibody (lanes 3 and 7), anti-p65 polyclonal antibody (lanes 4 and 8),anti-C/EBP polyclonal antibody (lanes and 9) or without antibody (“−”,lanes 1, 2 and 6); BALB/C (FIG. 9A), NOD (FIG. 9B). The labeled DNAprobe was then added to the reaction mixture. Again, a control reactionto which no nuclear extract was added was run in lane 1 as a negativecontrol. In all panels, DNA/NFκB complexes (NF-κB) and super-shiftedDNA-protein complexes (S-NF-κB) were indicated by arrows. Nuclearextracts were prepared from males (M) and females (F).

As in previous experiments, the prominent finding is that inTNF-α-stimulated Balb/c mice, the nucleus possesses an abundance of theactive form of NFκB (i.e., p50), as demonstrated by supershift. Incontrast, the NOD mouse appears unresponsive for p50 activation, evenafter exposure to a stimulant of NFκB activation.

Aberrant p52 proteins are found in lymphocytes, as a result ofchromosome rearrangements at the NFKB2 locus (Neri et al., 1991, Cell,67: 1075), p52 is normally produced as p100, an inactive precursorharboring IκB-like ankyrincontaining sequences in its C-terminal halfand presumably similarly processed by the proteasome. To demonstratewhether p52 binds κB oligonucleotide probe, supershift assays bypolyclonal antibody to p52 were carried out. Nuclear extracts wereincubated in the absence (−) or presence (+) of polyclonal antibodies top52 before EMSA with a κB binding sequence oligonucleotide probe.Original DNA-protein complexes (NF-κB) and supershifted DNA-proteincomplexes (S-NF-κB) are indicated by arrows. In supershift analysisperformed with the nuclear extracts of TNF-α-treated spleen cells,anti-p52 polyclonal antibody had no effect on the DNA-protein complexesin the nuclear extracts prepared from TNF-α-treated spleen cells, bothBALB/c and NOD (FIG. 9C).

The basal expression of NF-κB subunits, IκBa and cyclin-dependentkinases was examined by immunoblot analysis of cytosolic and nuclearextracts of male (M) or female (F) BALB/c and NOD mouse spleen cells(FIG. 10). In these experiments, extracts of spleen cells were subjectedto SDS-PAGE on a 12.5% gel under non-reducing conditions. The separatedproteins were transferred electrophoretically to a polyvinylidenedifluoride (PVDF) membrane which was then incubated for 2 hours at roomtemperature with TBS-T (20 mM Tris-HCl, pH 7.6; 137 mM NaCl; 0.05%volume/volume Tween 20) containing 8% (weight/volume) bovine serumalbumin. The membrane was then incubated for 12 h at 4° C. with TBS-Tcontaining the appropriate polyclonal antibodies, washed 4×5 minuteswith TBS-T at room temperature, incubated for 2 hours at roomtemperature with TBS-T containing alkaline phosphatase-conjugatedsecondary antibodies, washed five times with TBS-T and subjected to thealkaline phosphatase color reaction. In cytosolic extracts, theabundance of p65 and precursor p105, as well as that of thecyclin-dependent kinases CDK8, CDK7, and CDK2 (assayed as controls), didnot differ between BALB/c and NOD mouse spleen cells. The expression ofp50 in cytosolic extracts from spleen cells from NOD mice (male andfemale) was, however, markedly reduced relative to that in those fromBALB/c mice. In nuclear extracts, basal expression levels of p65 weresimilar in the two mouse strains, the expression of p50 was virtuallyinapparent in NOD mice. Furthermore, the basal expression of p52 incytoplasmic and nuclear extracts of BALB/c and NOD mouse spleen cellswas examined by immunoblot analysis. In cytosolic extracts, the basalexpression of precursor p100, as well as that of the cyclin-dependentkinases and p65(RelA), did not differ in spleen cells from BALB/c andNOD (male and female). However, the basal expression of p52 in thecytosolic extracts from NOD mice spleen cells was significantly reducedrelative to BALB/c mice (FIG. 10A). Northern blot analysis also revealedthat the abundance of both p65 and p 1 05 mRNAs in cytosolic extracts ofspleen (or lung) cells did not differ between BALB/c and NOD mice (datanot shown).

To assess the dynamics of IκBα protein expression occurring duringlymphocyte activation by TNF-α treatment, subcellular fractions fromlymphocytes derived from BALB/c and NOD spleen were treated with 10ng/ml TNF-α. Fractions were collected for preparation of cytosolicextracts at the indicated times and then subjected to immunoblottingwith appropriate antibodies. IκBα protein was readily detected in thecytosolic extracts from both unstimulated lymphocytes, BALB/c and NOD.In BALB/c lymphocytes treated by TNF-α, the cytosolic IκBα disappearedwithin 40 minutes of stimulation without concomitant expression in thenucleus; furthermore IκBα protein reappeared in the cytoplasm after 4hours of stimulation (FIG. 10B). In NOD lymphocytes, however, cytosolicIκBα was clearly detected after 40 minutes of stimulation and thenstably expressed during TNF-α treatment. This finding indicates a likelydefect in the proteasome degradation of IκBα in TNF-α-treatedlymphocytes from NOD mice (FIG. 10B).

The processing of p105 to p50 is mediated by the proteasome processingpathway (Fan and Maniatis, 1991, Nature, 354; 395; Maniatis, 1997,Science, 278: 818; Scherer et al., 1995, Proc. Natl. Acad. Sci. U.S.A.,92: 11258; Palombella et al., 1994, Cell, 78: 773; Coux and Goldberg,1998, J. Biol. Chem., 273: 8820; Sears et al., 1998, J. Biol. Chem.,273: 1409; Pahl and Baeuerle, 1996, Curr. Opin. Cell Biol., 8: 340).Proteasome inhibitors block activation of NF-κB and reduce cell survivalafter exposure to TNF-α (Cui et al., 1997, Proc. Natl. Acad. Sci.U.S.A., 94: 7515). It is possible that proteasome dysfunction in NODmice is attributable in part to down-regulation of LMP2, lone of the βsubunits of the 20S proteasome (Yan et al., 1997, Immunol., 159: 3068).LMP2 is thought to be required for the biological activity of the 20Sproteasome (Schmidtke et al., 1996, EMBO J., 15: 6887).; Schmidt andKloetzel, 1997, FASEB J., 11: 1235).

Furthermore, in the T12 cell line, in which Lmp2 and Lmp7 genes havebeen deleted, NF-κB is not activated in response to TNF-α. The effect ofTNF-α treatment on the DNA-binding activity of nuclear NF-κB wasexamined (FIG. 11). EMSA was performed such that cell extracts frommutant T2 cells compared to those of control T1 cells, Molt-4 cells andJurkat cells after stimulation for 4 h in the absence (−) or presence(+) of 10 ng/ml TNF-α. Lane 1 corresponds to a negative control in whichno nuclear extract was added to the reaction mixture (arrowheadindicates specific DNA-protein complexes). In TNF-α treated-cell linesT1 cells, Molt-4 cells and Jurkat cells, the expression of the activenuclear form of NF-κB was markedly detected on EMSA; however NF-κBactivity was not induced in TNF-α-treated T2 cells (FIG. 11A).

The specificity of DNA-binding activity in the nuclear extracts preparedfrom these cell lines was confirmed by preincubation of these nuclearextracts in the presence (+) or absence (−) of a 100-fold molar excessof unlabeled competitor oligonucleotide comprising a wild-type κBbinding site (w), a mutant site, κB1 (m1) or a second mutant site, κB2(m2) before addition of ³

-unlabeled oligonucleotide (κB1). Double-stranded oligodeoxynucleotideswere synthesized on a DNA synthesizer by the phosphoramidite method andpurified on an OPC cartridge. They corresponded to κB binding motifs ofthe human immunodeficiency virus-type 1 enhancer (5′-GATCTAGGGACTTTCCGCTGGGGACTTTCCAG-3′; κB1(SEQ ID NO:1) and interleukin-2receptor a chain gene enhancer (5′-GAT CTCAGGGGAATCTCCCTCTCCTTTTATGGGCGTAGCG-3′; κB2(SEQ ID NO:2). The oligonucleotides wereend-labeled with [α-³²P]dCTP and Klenow polymerase. Nuclear extract wasincubated at 37° C. for minutes in a total volume of 10 μl containing 10mM Hepes-NaOH (pH 7.9), 50 mM KCl, 5 mM Tris-HCl (pH 7.0), 1 mM DTT, 15mM EDTA, 10% (v/v) glycerol, 1.0 μg of poly (dI.dC) and 4 ng of³²P-labeled κB oligonucleotide. The DNA-protein complexes were resolvedby electrophoresis on nondenaturing 8% polyacrylamide gels with 0.5×TBE(Tris-borate-EDTA) buffer at 4° C. For competition experiments, nuclearextracts was incubated for minutes at 4° C. with a 100-fold molar excessof unlabeled κB oligonucleotide before addition of the radioactiveprobe. Cytosolic extracts were treated with 1.2% NP-40 and 0.8%deoxycholate to dissociate IκB from NF-κB before incubation with3²P-labeled probe. For supershift assays, nuclear extracts wereincubated with specific antibodies in 1 hour at 4° C. before addition ofDNA probes. Lanes 1 correspondes to negative controls in which nuclearextract was not added.

NF-κB DNA-binding activity in the cytosolic extracts was analyzed byEMSA with the κB1 oligonucleotide after pre-incubation with (+) orwithout (−) NP-40 and deoxycholate (FIG. 11C). Lane 1 corresponds to anegative control in which cytosolic extract was not added to thereaction mixture. The κB binding activity in the cytsolic extractsprepared from T2 cells was dramatically reduced relative to thatapparent in other cytoplasmic extracts (FIG. 11C).

The specificity of the κB DNA- binding activity in T2 cells and theextract quality were confirmed by the DNA-binding activities of theother transcription factors, SP1 and AP1 on EMSA with specificoligonucleotide probes (FIG. 11D). DNA-binding activities of SP1 (left)or AP1 (right) in the nuclear extracts of these cell lines were measuredand were found not to differ among extracts from the T1, T2, Jurkat andMolt-4 cell lines (FIG. 11D).

Supershift assays were performed with the nuclear extracts prepared fromT1 and T2 cells (FIG. 11E, top) or Molt-4 and Jurkat cells (FIG. 11E,top). Nuclear extracts prepared from these cell lines were incubated inthe absence (−) or presence (+) of polyclonal antibodies to p50 (lanes3, 7), to p65 (lanes 4, 8), or to C/EBP (lanes 5, 9) before EMSA withthe κB1 oligonucleotide. Non-shifted DNA-protein complexes (NF-κB) andsupershifted DNA-protein complexes (S-NF-κB) are indicated by arrows. Inthese experiments, antibodies to p50 or to p65 shifted the bands of theDNA-protein complexes in all nuclear extracts of T1 cells, Molt-4 cellsand Jurkat cells on the EMSA, while no supershift band was detected inthe T12 cells nuclear extract pre-incubated with the anti-p50 antibodies(FIG. 11E). Antibodies to C/EBP had no effect on the DNA-proteincomplexes in all nuclear extracts of these cell lines (FIG. 11E).

The basal expression levels of NF-κB subunits, IκBα and cyclin-dependentkinases were determined by immunoblot analysis of cytosolic and nuclearextracts of T1, T2, Molt-4 and Jurkat cells. Cytosolic and nuclearextracts prepared from these cell lines were subjected to immunoblotanalysis with the appropriate antibodies, as described above. Incytosolic extracts, the basal expression of p65, precursor p100 andp105, as well as that of the cyclin-dependent kinases, did not differ inthese cell lines; however, the expression of p50 and p52 in cytoplasmicextracts prepared from T2 cells was significantly reduced relative tothat in those from other cell lines (FIG. 11F). In nuclear extracts,although the basal expression levels of p65 were similar in the thesecell lines, the expression of neither p50 nor p52 was clearly detectedin T2 cells (FIG. 11F). The findings presented in FIG. 11 suggest thatspecific proteasome subunits are required for the activation of NF-κB byTNF-α treatment.

To investigate whether the altered abundance of p50 in NOD mouse spleencells could be attributable to defective processing of p105 by theproteasome, the processing of p105 by cytosolic extracts of NOD mousespleen cells was examined using recombinant p105 or the truncatedversion p60Tth as substrates. The in vitro p105 processing assayreaction was performed as previously described (Fan and Maniatis, 1991,supra). In brief, p105 and p60Tth expression constructs were subjectedto in vitro transcription and translation in a wheat germ extract system(Promega) in the presence of [³⁵S]methionine. The ³⁵S-labeled p105 andp60th proteins were immunoprecipitated with polyclonal antibodies to p50and purified for use as substrates. Each substrate protein was incubatedfor 90 minutes at 30° C. with spleen cytosolic extract (20 or40 μg ofprotein) in a final volume of 25 μl in the absence or presence of 10 mMATP (Palombella et al., 1994, supra). The proteasome inhibitor MG115 wasalso added to the reaction mixture where indicated (FIG. 12). Theprocessed proteins were separated by SDS-polyacrylamide gelelectrophoresis (PAGE) on a 10% gel and visualized by autoradiography.Incubation of p60Tth with cytosolic extracts of neither BALB/c nor NODmouse spleen cells resulted in the generation of the cleaved fragment inthe absence of ATP (FIG. 12A, left). When p60Tth was incubated withcytosolic extracts of BALB/c cells in the presence of mM ATP, mature p50was generated (FIG. 12A, center). Although p60Tth was incubated withcytosolic extracts of NOD cells in the presence of 10 mM ATP, p50 wasnot produced (FIG. 12A, center). The production of p50 has previouslybeen shown to be stimulated by ATP (Fan and Maniatis, 1991, supra;Palombella et al., 1994, supra).

Similar results were obtained when p105 was used as substrate, althoughthe extent of processing was less than that observed with p60Tth (FIG.12B). ATP-dependent processing of both p60Tth and p105 with cytosolicextracts of NOD mouse spleen cells was clearly impaired and the defectappeared more pronounced for NOD females than for NOD males (FIGS. 12Aand 12B). To confirm that the formation of p50 in this in vitro assaywas mediated by the proteasome, we the effect of MG115 on proteasomefunction was examined. MG115 is a potent inhibitor of the chymotrypticsite on the 20S proteasome particle, and has previously been shown toreduce the degradation of ubiquitin-conjugated proteins in cell extractsand, at a concentration of 50 μM, to prevent the processing of p105(Palombella et al., 1994, supra). In the present study, the processingof p105 and p60Tth was also completely inhibited by MG115 at aconcentration of 50 μM (FIGS. 12A, right and 12B, right).

A PEST-rich domain downstream of the ankyrin repeats of p105 isphosphorylated after stimulation, but the phosphorylation of thec-terminus of p105 produces no clear functional consequences (Sears etal., 1998, supra; Lin et al., 1998, Cell, 92: 819; Naumann andScheidereit, 1994, EMBO J., 13: 711; Pahl and Baeuerle, 1996, supra;MacKichan et al., 1996, J. Biol. Chem., 271: 6084; Fujimoto et al.,1995, Gene, 165: 183). The phosphorylation status of recombinant p105was examined in cytosolic extracts of spleen cells from BALB/c and NODmice (FIG. 12C). To do this, recombinant p105 was incubated for varioustimes at 30° C. in a reaction mixture containing [γ-32P]ATP andcytosolic extracts (40 μg of protein) of spleen cells from male orfemale BALB/c or NOD mice, after which p 105 was immunoprecipitated withantibodies to p50 and subjected to SDS-PAGE and autoradiography. Thepositions of phosphorylated p105 and of p50 are indicated (FIG. 12C).Phosphorylation of p105 by cytosolic extracts of BALB/c spleen cellsreached a maximum at 30 minutes and thereafter decreased, presumablybecause the phosphorylated protein was degraded by theubiquitin-proteasome pathway (FIG. 12C). In contrast, thephosphorylation of p105 by cytosolic extracts of spleen cells from NODmice (male and female) continued to increase for up to 40 minutes,presumably because the phosphorylated protein did not undergoproteolysis (FIG. 12C). Thus, the activity of the p105 kinase appearsnormal in cytosolic extracts of NOD mouse spleen cells.

Ubiquitination of the ankyrin repeats of p105 is also thought in mostcases to be required for its proteolytic processing (Palombella et al.,1994, supra; Coux and Goldberg, 1998, supra; Sears et al., 1998, supra;Pahl and Baeuerle, 1996, supra). The ubiquitination of p105 was examinedafter incubation of the protein with cytosolic extracts of BALB/c andNOD mouse spleen cells (FIG. 12D). Recombinant p105 was incubated forvarious times at 30° C. in a reaction mixture containing cytosolicextracts (40 μg of protein) of spleen cells from male or female BALB/cor NOD mice, after which complexes were cross-linked withglutaraldehyde, immunoprecipated with antibodies to p50, and detected byimmunoblot analysis with antibodies to ubiquitin. The positions ofubiquitinated p105 (ubn-p105) and of molecular size standards (inkilodaltons) are indicated. Impaired NF-κB activity in the NOD mouse dueto defective processing by the proteasome. Cross-linking of ubiquitinp105 complexes with glutaraldehyde, followed by theirimmunoprecipitation by antibodies to p50 and immunoblot analysis withantibody to ubiquitin, revealed a temporal pattern for ubiquitinationsimilar to that for phosphorylation of p105. Thus, whereas theubiqutination of p105 by cytosolic extracts of BALB/c cells reached amaximum at 30 minutes and thereafter decreased, that mediated byextracts of NOD mouse (male and female) cells continued to increase forup to 40 minutes (FIG. 12D). Thus, ubiquitination activity appeared notto be down-regulated in cytosolic extracts of NOD mouse spleen cells.Overall, these data localize the defect in p105 processing in NOD mousecells to the proteasome.

These results suggest that the activity of the proteasome particle ofNOD mouse cells is impaired with regard to p105 processing. Thisimpaired p105 proteolytic processing is also consistent with therelative toxicity of TNF-α in NOD spleen cells.

To demonstrate the defective proteosome processing pathway of p105 in T2cells, the processing of p105 in cytoplasmic extracts of T1 cells, T2cells, Molt-4 cells and Jurkat cells was investigated by in vitro assaywith ³⁵S-labeled and purified recombinant p105 as substrate. The labeledp105 was incubated with the cytosolic extracts of these cell lines (20or 40 μg of protein at left and center) and the reaction mixtures wereincubated with (center; FIGS. 12E, F and G) or without (upper; FIGS.12E, F and G) 10 mM ATP in a wheat germ extract system (Promega) asabove. Incubations were also performed in the absence (−) or presence(+) of 50 μg MG115 (lower; FIGS. 12E, F and G). Lane 1 in all gelspresented in FIGS. 12E, F and G corresponds to reaction mixtures withoutsubstrate. Incubations were performed at 30° C. for 90 minutes, afterwhich the reaction mixtures were analyzed by SDS-PAGE andautoradiography. Incubation of p105 with cytoplasmic extracts preparedfrom these cell lines in the absence of ATP did not result in thegeneration of the cleaved fragment p50(FIG. 12E). When p105 wasincubated with the cytoplasmic extracts of T1 cells, Molt-4 cells andJurkat cells in the presence of 10 mM ATP, the mature p50 was detected(FIG. 12F); however, the p50 was not generated by incubation of p105with the cytoplasmic extracts prepared from T2 cells in the presence of10 mM ATP (FIG. 12F, lanes 4 and 5). To verify that the maturationprocessing of p50 in the in vitro reaction was mediated by theproteasome processing pathway, the effect of MG115 on proteasomefunction was examined. Addition of MG115 into the reaction mixtureclearly resulted in defective p105 processing (FIG. 12G).

To determine the basal expression level of components of the 20Sproteasome and cyclin-dependent kinases in the T1, T2, Molt-4 and Jurkatcell lines, the immunoblot analysis was performed with appropriateantibodies on cytosolic and nuclear extracts. The basal expression levelof proteasome components were also compared to TAFII250 andcyclin-dependent kinases (CDK2, CDK7 and CDK8). The results demonstratedthat the basal expression of TAFII250 and cyclin-dependent kinases didnot differ among these cell lines (FIG. 12H). In the case of proteasomecomponents, the lack of expression of LMP2 and LMP7 in the cytoplasmicextracts prepared from T2 cells was confirmed by immunoblot analysis(FIG. 12H).

One critical function of NFκB is to provide protection to cells from theeffects of exogenous TNF-α. In the experiments shown in FIG. 13A, spleencells were prepared from BALB/c and NOD mice, and tested for survivalfollowing TNF-α stimulation. Spleen cells were cultured for 24 hoursafter exposure to various concentrations (2, 5, 10 or 20 ng/ml) ofTNF-α, as indicated on the X axis of the figure. Viable cells remainingafter TNF-α treatment are shown as a percentage of viable control(untreated) cells. Standard deviations were calculated from fourindependent readings within a single experiment. The survival over timeof cells treated with TNF-α is charted in FIG. 13B. Spleen cells weretreated with TNF-α (10 ng/ml), and viable cells were counted at varioustimes following treatment as indicated on the X axis of the figure.

These data clearly demonstrate that TNF-α treatment is toxic to NOD miceand the cells experience rapid death. That the survival of NOD mice iscompromised with regard to that of normal mice indicates clearly thatNFκB activation is defective in this autoimmune mouse model.

DNA fragmentation was evaluated and detected by agarose gelelectrophoresis after spleen cells were cultured in 10 ng/ml TNF-α for24 hours. These assays confirmed TNF-α treatment of NOD but not BALB/cspleen cells resulted in apoptosis as demonstrated by agarose gelelectrophoresis (FIG. 13C). Embryonic fibroblasts prepared from BALB/cand NOD mice were cultured DMEM containing 10% fetal bovine serum andthen incubated with various concentrations of TNF-α for 24 hours (FIG.13D, top) or with TNF-α at 10 ng/ml for the indicated times (FIG. 13D,bottom). Cell viability was assessed by trypan blue exclusion. Data aremeans±SD of four replicates from a representative experiment, and areexpressed as a percentage of the survival value for the correspondingcells not exposed to TNF-α. In contrast to the data of FIG. 13C,recently established cultures of NOD mouse embryonic fibroblasts (MEF)demonstrated no cellular toxicity with TNF-α exposure suggesting tissueor developmental specificity of the NF-κB defect (FIG. 13D).

The promoter of the Lmp2 gene in the NOD mouse contains a candidatemutation that may reduce transcription and/or translation (Yan et al.,1997, J. Immunol., 159: 3068). NOD mice have reduced Lmp2 mRNA inlymphocytes and reduced reporter protein in transcient transfectionassays. To determine at the protein level whether the NOD mouse exhibitslow in vivo basal protein expression of the LMP2, LMP7, LMP10 or C9components of the 20S proteasome, immunoblot analysis was conducted onsplenic and mouse embryonic fibroblasts (MEF) extracts. Basal expressionof TAFII250 and cyclin dependent kinases CDK2, CDK7, and CDK8 werecompared to basal expression levels of proteasome components byimmunoblot in spleen cells (extracts from male and female BALB/c and NODmice) (FIG. 14A) and mouse embryonic fibroblasts (MEF) derived fromBALB/c and NOD mice (FIG. 14B). Purified antibodies were used to detectTAFII250, CDK2, CDK7, and CDK8 as well as p105, p50 and p65 of NF-κB andc-Rel under conditions as described above. Anti-sera recognized murineLMP2, LMP7 and LMP10 proteasome components and C9 antibody recognizedboth proteasome precursors as well as mature proteasomes. The basalexpression level of proteasome protein was also compared to TAFII250,which is a factor that promotes cell cycle progression, and to cyclindependent kinases CDK2, CDK7 and CDK8.

TNF-α stimulated spleen cells were evaluated for downstream c-mycprotein, a transcriptionally-induced protein by properly activated NF-κBsubunits. These data show, using polyclonal antibody detection methods,that basal expression levels of TAFII250, CDK2, CDK7, and CDK8 areequivalent for spleen cells of male and female mice from both the NODand BALB/c strains (FIG. 14A). The basal expression levels of NF-κBsubunits p50, p65, p105 precursor and c-Rel were examined in bothcytosolic and nuclear extracts of BALB/c and NOD mice spleen cells byimmunoblot analysis (FIG. 14A). The expression of p50 in cytosolic andnuclear extracts from spleen cells derived from NOD mice wassignificantly lower than that observed in BALB/c mice (FIG. 14A). In thecase of proteasome proteins, the NOD mice lacked detectable basalexpression of LMP2 selectivity in spleen cells but not MEF (FIGS. 14Aand 14B). NOD spleen cells and MEF both exhibited normal levels of LMP7,LMP10 and the C9 proteasome subunits. The C9 antibody recognizes mostprecursor proteasomes and mature proteasomes (Nandi et al., 1997, EMBOJ., 16: 5363). Furthermore, MEF from NOD mice expressed normal levels ofboth NF-κB subunits p50, p65, p105 precursors and c-Rel in whole celllysates (FIG. 14B). FIG. 14 indicates that transcriptional activatedheterodimer complexes, p50-p65 and p50-c-Rel were impaired in NOD micespleen cells but not MEF. These findings suggest that Rel/NF-κBdysfunction as positive transcriptional regulators for severalinterleukins and their receptors, the c-myc proto-oncogene and a varietyof adhesion molecules in NOD lymphoid cells. In addition, expression ofthe gene encoding proto-oncogene product c-myc was not strongly inducedin TNF-α treated NOD spleen cells (data not shown). NF-κB dysfunctionwas apparent in TNF-α induced NOD spleen cells by the total lack ofc-myc protein (data not shown).

While the data presented above indicate that the phosphorylation andubiquitination of p105 appears normal in NOD mouse spleen cells, it wasof interest to determine whether the proteolytic processing of p105 top50 by the proteasome is impaired in these animals (FIG. 15). The mutantT2 cell line, deficient in MHC-encoded LMP protein in a mannercomparable the NOD mouse, was seen to experimentally mirror NODsplenocytes for deranged NF-κB activation and downstream nuclear events.The markedly defective function of the proteasome in NOD mouse spleencells was associated with impaired TNF-α induced NF-κB activation andincreased susceptibility to TNF-α induced apoptosis. The proteasomecutting detect extended to defective p100 processing to p52 subunits aswell as interrupted IκB-α degradation, indicating that NOD mouse spleencells have an immature proteasome in which processing of p105 to thep50subunit is blocked.

The results above indicate that the protein complexes observed in NODmice differ from those seen in mice of the BALB/c strain, prompting amore rigorous examination of the DNA binding specificity. In particular,mutant oligonucleotides which are not bound by the active p50/p65complex were tested as specificity controls. As reported in thepublished literature, the p50-p65 heterodimer interacts with artificialpalindromic κB binding motifs which duplicate the half sites in themotif 5′-GGGACTTTC-3′(SEQ ID NO:4)AAB) into 5′-GGGACGTCCC-3′(SEQ IDNO:5(AA) and 5′-GAAATTTCC-3′(SEQ ID NO:6)(BB) (Urban and Baueurle, 1990;Urban et al., 1991). A published competition assay revealed that activep50-p65 heterodimer was unable to bind the two palindromic sites, AA andBE with high affinity (Urban and Baueurle, 1990, supra; Urban et al.,1991, supra).

To verify the impairment of the p50-p65 active form in NOD lymphocytes,κB site binding protein was tested in a competition assay with ³⁷P-labeled AB probe, using unlabeled (SEQ ID NO:4) AB, AA(SEQ ID NO:5)and BB(SEQ ID NO:6) oligonucleotides as competitors. In TNF-α-treatedBALB/c lymphocytes, κB site binding protein was unable to bind twopalindromic sites AA (SEQ ID NO:5) and BB (SEQ ID NO:6) affinityindicating that the nuclear form of p50-p65 had been induced. Incontrast, κB site binding protein was able to bind the BBoligonucleotides with high affinity inthe TNF-α-treated NOD lymphocytes(FIG. 16). This assay, using competitive oligonucleotides, indicatesdefective nuclear expression of p50-p65 active forms in TNF-α-treatedNOD lymphocytes.,

USE

The invention is of use in the diagnosis and treatment of autoimmunedisorders

OTHER EMBODIMENTS

Other embodiments will be evident to those of skill in the art. Itshould be understood that the foregoing description is provided forclarity only and is merely exemplary. The spirit and scope of thepresent invention are not limited to the above examples, but areencompassed by the following claims.

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What is claimed is:
 1. A method of restoring NFκB activity in a mammalafflicted with an automimmune disease resulting from a reduction in NFκBactivity, comprising administering to a mammal suspected of sufferingfrom said autoimmune disease a therapeutically effective amount of aprotein which restores NFκB activity so as to treat said disease in saidmammal.
 2. The method according to claim 1, wherein said protein isselected from the group consisting of: NFκB, NFκB p50, NFκB p52, NFκBp65 and IκB.
 3. The method according to claim 1, wherein said mammal isa human.
 4. The method according to claim 3, wherein said autoimmunedisease is an HLA class II-linked disease.